Using Targeted Radiotherapy (TRT) to Drive Anti-Tumor Immune Response to Immunotherapies

ABSTRACT

The disclosed method of treating a malignant solid tumor in a subject includes the steps of administering to the subject an immunomodulatory dose of a radioactive phospholipid ether metal chelate, a radiohalogenated phospholipid ether, or other targeted radiotherapy (TRT) agent that is differentially retained within malignant solid tumor tissue, and either (a) performing in situ tumor vaccination in the subject by introducing into at least one of the malignant solid tumors one or more agents capable of stimulating specific immune cells within the tumor microenvironment, or (b) performing immunotherapy in the subject by systemically administering to the subject an immunostimulatory agent, such as an immune checkpoint inhibitor. In a non-limiting example, the radioactive phospholipid ether metal chelate or radiohalogenated phospholipid ether has the formula:wherein R1 comprises a chelating agent that is chelated to a metal atom, wherein the metal atom is an alpha, beta or Auger emitting metal isotope with a half-life of greater than 6 hours and less than 30 days, or wherein R1 comprises a radioactive halogen isotope. In one such embodiment, a is 1, n is 18, m is 0, b is 1, and R2 is —N+(CH3)3.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/809,427, filed on Nov. 10, 2017, now U.S. Pat. No. 11,635,506, whichis a continuation-in-part of U.S. application Ser. No. 15/652,400, filedon Jul. 18, 2017, now U.S. Pat. No. 10,736,949, which claims the benefitof U.S. Provisional Application No. 62/363,608, filed on Jul. 18, 2016;and U.S. application Ser. No. 15/809,427 is a continuation-in-part ofU.S. application Ser. No. 15/658,535, filed on Jul. 25, 2017, now U.S.Pat. No. 10,751,430, which claims the benefit of U.S. ProvisionalApplication No. 62/366,340, filed Jul. 25, 2016. Each of theseapplications is incorporated by reference herein in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under OD024576 andCA197078 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

FIELD OF THE DISCLOSURE

This disclosure relates generally to methods of treating cancer. Inparticular, the disclosure is directed to methods of treating a cancercomprising one or more malignant solid tumors in a subject by (1)systemically administering to the subject an immunomodulatory dose of atargeted radiotherapy (TRT) agent, such as a radioactive metal chelatecompound, a radiohalogenated compound, radiolabeled antibody, or aradioisotope that is differentially taken up by and retained withinsolid tumor tissue, and (2) either (a) performing in situ tumorvaccination in the subject at one or more of the malignant solid tumorsusing one or more locally-administered treatments capable of stimulatingspecific immune cells within the tumor microenvironment, or (b)systemically administering to the subject one or more immunostimulatoryagents, such as one or more immune checkpoint inhibitors.

BACKGROUND

Current cancer treatment typically involves systemic chemotherapywhereby non-targeted small molecule or antibody directed cytotoxicagents preferentially enter, or bind to (in the case of antibodydirected agents) and kill cancer cells by a variety of mechanisms.External beam radiation therapy (xRT), which is often combined withchemotherapy, kills cancer cells by inducing nuclear DNA double strandbreaks resulting in cell-cycle death. Unlike systemic chemotherapy, xRTdepends on the ability to accurately determine the anatomic location ofthe tumor. Surgical resection of tumors also depends on the ability tosee the tumor and on complete removal, since residual tumor cells willquickly reestablish the tumor following surgery. Surgery and xRT aregenerally limited to the local treatment of malignant tumors and thusare limited in treating disseminated or metastatic disease, which is whychemotherapy is often used in conjunction with these treatmentmodalities. Although systemic chemotherapy is capable of reaching manydistant metastatic sites, with the possible exception of brainmetastases, for all too many patients, responses are typicallyshort-lived (months to several years) and ultimately result in tumorrecurrence.

Because the body's natural immune system is also capable of destroyingcancer cells following their recognition, immunologic approaches arerapidly becoming more prevalent in cancer treatment paradigms. However,some cancer cells, and to a greater extent cancer stem cells, manage toinitially avoid immune-surveillance and actually acquire the ability toevolve and ultimately survive by remaining relatively immune invisible[Gaipi et al, Immunotherapy 6:597-610, 2014].

One specific immunologic approach that is being increasinglyinvestigated is “in situ vaccination,” a strategy that seeks to enhancetumor immunogenicity, generate tumor infiltrating lymphocytes (TIL) anddrive a systemic anti-tumor immune response directed against“unvaccinated,” disseminated tumors. In in situ vaccination, a malignantsolid tumor is injected with (or treated with) one or more agents thatfacilitate the release of tumor antigens while simultaneously providingpro-inflammatory signals to reverse the immune-tolerizingmicroenvironment of the tumor [Pierce et al, Human Vaccines &Immunotherapeutics 11 (8):1901-1909, 2015; Marabelle et al, Clin. CancerRes. 20(7):1747-56, 2014; Morris et al, Cancer Res; ⁷6(13); 3929-41,2016].

A second and quite different approach is systemically-administeredimmunotherapy. In systemically-administered immunotherapy, animmunostimulatory agent, such as an immune checkpoint inhibitor, isadministered to circulate through the entire body (e.g., intravenously),rather than being locally injected into the tumor. Such agents can beused to treat tumors in which an anti-tumor immune response is present,but has been “exhausted” or rendered ineffective. In the case ofcheckpoint inhibitors, the tumor cells express “checkpoint ligands” orother checkpoint molecules that interact with “checkpoint receptors” onthe existing anti-tumor immune cells, triggering the inactivation ofthese cells. By blocking this interaction, systemically-administeredcheckpoint inhibitors turn on the exhausted, pre-existing immuneresponse in cancer patients, facilitating a more effective attack on thecancer cells by the patient's own immune system.

Although recent data from clinical trials and pre-clinical modelsillustrate the potential of these approaches, there is a great need inthe art for in-situ vaccination or systemically-administeredimmunotherapy methods exhibiting improved systemic efficacy.

Radiation hormesis is a decades-old hypothesis that low doses ofionizing RT can be beneficial by stimulating the activation of naturalprotective repair mechanisms that are not activated in the absence ofionizing RT [Cameron and Moulder, Med. Phys. 25:1407, 1998]. The reserverepair mechanisms are hypothesized to be sufficiently effective whenstimulated as to not only cancel the detrimental effects of ionizing RTbut also inhibit disease not related to RT exposure. Perhaps related,the abscopal effect is a phenomenon reported in the 1950's, whereby, xRTtreatment of one tumor actually causes shrinkage of another tumoroutside the RT treatment area. Although rare, this phenomenon is thoughtto be dependent on activation of the immune system. Together, hormesisand the abscopal effect support the potential interaction andstimulation of the immune system by low dosage (immune stimulatory butnon-cytotoxic) RT, which may then be combined with other immunologicapproaches, such as in situ vaccination or systemically-administeredimmunotherapy.

We have previously published that the combination of local xRT+ in situvaccination and/or systemic checkpoint inhibitor immunotherapy arepotently synergistic in treating large established tumors in mice, whenthere is a single tumor present [Morris et al, Cancer Res; 76(13);3929-41, 2016]. However, we have surprisingly discovered (and discloseherein) that the combination of in situ vaccination and xRT does notresult in inhibited tumor growth in the presence of a second,non-radiated tumor. Apparently, the non-radiated tumor exhibits adampening effect (which we have designated as “concomitant immunetolerance”) on the immunomodulatory effect of the xRT and in situvaccine on the radiated tumor.

This concomitant immune tolerance can be overcome, enabling efficacy ofin situ vaccination, when xRT is given to all areas of tumor. However,xRT cannot be effectively used in combination with in situ vaccinationmethods in the presence of multiple tumors, particularly if the tumorsare not few in number, or if the location of one or more of the tumorsis not precisely known, or if it is not feasible to deliver xRT to allsites of tumor. Furthermore, administering xRT to all tumor sites inpatients with metastatic disease would likely result in systemic immunesuppression, defeating the central purpose of systemically-administeredimmunotherapy.

Accordingly, in combination with either in situ vaccination orsystemically-administered immunotherapy, there is a need for improvedmethods of delivering an immunomodulatory dose of RT to all tumorswithin a subject, regardless of their number and anatomic location.

BRIEF SUMMARY

We have previously shown that certain alkylphosphocholine analogs arepreferentially taken up and retained by malignant solid tumor cells. InU.S. Patent Publication No. 2014/0030187, which is incorporated byreference herein in its entirety, Weichert et al. disclose using analogsof the base compound 18-(p-iodophenyl)octadecyl phosphocholine (NM404;see FIG. 1 ) for detecting and locating, as well as for treating, avariety of malignant solid tumors. If the iodo moiety is animaging-optimized radionuclide, such as iodine-124 ([¹²⁴I]-NM404), theanalog can be used in positron emission tomography-computed tomography(PET/CT) or single-photon emission computed tomography (SPECT) imagingof solid tumors. Alternatively, if the iodo moiety is a radionuclideoptimized for delivering therapeutic doses of RT to the solid tumorscells in which the analog is taken up, such as iodine-125 or iodine-131([¹²⁵I]-NM404 or [¹³¹I]-NM404), the analog can be used to treat thesolid tumors.

Such analogs not only target a wide variety of solid tumor types invivo, but also undergo prolonged selective retention in tumor cells,thus affording high potential as RT agents. Moreover, tumor uptake islimited to malignant cancer and not premalignant or benign lesions.

However, there are metal isotopes that have better properties foroptimized imaging and/or RT than the radioactive iodine isotopes used inthe previously disclosed alkylphosphocholine analogs. For example, as animaging isotope, I-124 suffers from poor positron output (only about 24%of the emissions are positrons), and it suffers further from aconfounding gamma emission (600 KeV), which actually interferes withnormal 511 KeV PET detection. Certain positron emitting metals havebetter imaging characteristics. As another example, as an RT isotope,I-131 produces other non-therapeutic emissions at other energies, whichadd undesired radiation dosimetry to neighboring normal tissue,including bone marrow. The beta particle range of I-131 is also quitelong, which contributes to off target toxicity. Several metallicradiotherapy isotopes offer a cleaner emission profile and shorterpathlength and thus less potential toxicity.

We have developed improved alkylphosphocholine analogs that include achelated radioactive metal isotope instead of a radioactive iodineisotope (see, e.g., U.S. patent application Ser. No. 15/343,604, whichis incorporated by reference herein in its entirety). The analogsinclude the same backbone as the previously disclosed radioiodinatedcompounds, so they are still selectively taken up and retained in tumorcells. However, the chelated radioactive metal isotope provides improvedemissions for imaging and/or radiotherapy applications. Such agents arewell suited for delivering a sub-cytotoxic but immunomodulatory dose ofionizing RT to all malignant tumors present within a subject, regardlessof whether their number and locations are known.

Accordingly, in a first aspect, the disclosure encompasses a method oftreating a cancer comprising one or more malignant solid tumors in asubject. The method includes the steps of. (a) administering to thesubject an immunomodulatory dose of a radioactive phospholipid metalchelate compound that is differentially taken up by and retained withinmalignant solid tumor tissue; and (b) performing in situ tumorvaccination in the subject at one or more of the malignant solid tumorsusing one or more treatments capable of stimulating specific immunecells within the tumor microenvironment. An “immunomodulatory dose” is alow or sub-cytotoxic RT dose of the targeted radiotherapy agent.Although NM404 is used in some of the examples below, other examples usethe phospholipid metal chelate compound NM600, which similarly targetssolid tumor tissue. For radiotherapy application, the radioactive metalchelated into the compound could include any alpha, beta, auger, and/orgamma emitting metal. The key feature is that targeted radiotherapyagent emits low or sub-cytotoxic RT doses that are not lethal to eitherthe cancer cells or the relevant immune cells.

In some embodiments, the one or more treatments capable of stimulatingspecific immune cells within the tumor microenvironment include treatingthe tumor with xRT. In some embodiments, the one or more treatmentscapable of stimulating specific immune cells within the tumormicroenvironment include intratumorally injecting into at least one theone of the malignant solid tumors a composition that includes one ormore agents capable of stimulating specific immune cells within thetumor microenvironment. In some embodiments, such agents can include animmunostimulatory monoclonal antibody, a pattern recognition receptoragonist, an immunostimulatory cytokine, an immune stimulatorynanoparticle, an oncolytic virus, or any combinations thereof.Non-limiting examples of immunostimulatory monoclonal antibodies thatcould be used include anti-GD2 antibodies, anti-CTLA-4 antibodies,anti-CD137 antibodies, anti-CD134 antibodies, anti-PD-1 antibodies,anti-KIR antibodies, anti-LAG-3 antibodies, anti-PD-L1 antibodies,anti-CD40 antibodies, or combinations thereof. In some embodiments, theimmunostimulatory monoclonal antibody is an antibody to a tumor-specificantigen. In some embodiments, the composition that includes one or moreimmunostimulatory monoclonal antibodies may also include interleukin-2(IL-2). In some embodiments, the anti-GD2 antibody that is used mayinclude hu14.18, and optionally, may further include IL-2 (i.e., afusion protein of the two).

In some embodiments, the immunostimulatory cytokine is IL-2,interleukin-12 (IL-12), interleukin-15 (IL-15), interleukin-21 (IL-21),or an interferon (IFN).

In some embodiments, the pattern recognition receptor agonist is anagonist of a toll-like receptor (TLR). Non-limiting examples of suchTLRs TLR include TLR-1, TLR-2, TLR-3, TLR-4, TLR-5, TLR-6, TLR-7, TLR-8,TLR-9, or TLR-10.

In some embodiments, the radioactive phospholipid metal chelate compoundhas the formula:

or a salt thereof. R₁ comprises a chelating agent that is chelated to ametal atom, wherein the metal atom is an alpha, beta or Auger emittingmetal isotope with a half-life of greater than 6 hours and less than 30days; a is 0 or 1; n is an integer from 12 to 30; m is 0 or 1; Y is —H,—OH, —COOH, —COOX, —OCOX, or —OX, wherein X is an alkyl or an aryl; R₂is —N⁺H₃, —N⁺H₂Z, —N⁺HZ₂, or —N⁺Z₃, wherein each Z is independently analkyl or an aroalkyl; and b is 1 or 2. Non-limiting examples of metalisotopes that could be used include Lu-177, Y-90, Ho-166, Re-186,Re-188, Cu-67, Au-199, Rh-105, Ra-223, Ac-225, Pb-212, or Th-227.

In some embodiments, the chelating agent is1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3A) or one of itsderivatives; 1,4,7-triazacyclononane-1,4-diacetic acid (NODA) or one ofits derivatives; 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) orone of its derivatives;1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) or oneof its derivatives; 1,4,7-triazacyclononane,1-glutaric acid-4,7-diaceticacid (NODAGA) or one of its derivatives;1,4,7,10-tetraazacyclodecane,1-glutaric acid-4,7,10-triacetic acid(DOTAGA) or one of its derivatives;1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA) orone of its derivatives;1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4,11-diacetic acid (CB-TE2A)or one of its derivatives; diethylene triamine pentaacetic acid (DTPA),its diester, or one of its derivatives; 2-cyclohexyl diethylene triaminepentaacetic acid (CHX-A″-DTPA) or one of its derivatives; deforoxamine(DFO) or one of its derivatives;1,2-[[6-carboxypyridin-2-yl]methylamino]ethane (H₂dedpa) or one of itsderivatives; and DADA or one of its derivatives, wherein DADA comprisesthe structure:

In some embodiments, a is 1 (aliphatic aryl-alkyl chain). In otherembodiments, a is 0 (aliphatic alkyl chain).

In some embodiments, m is 1 (acylphospholipid series). In some suchembodiments, n is an integer between 12 and 20. In some embodiments, Yis —OCOX, —COOX or —OX.

In some embodiments, X is —CH₂CH₃ or —CH₃.

In some embodiments, m is 0 (alkylphospholipid series).

In some embodiments, b is 1.

In some embodiments, n is 18.

In some embodiments, R₂ is —N⁺Z₃. In some such embodiments, each Z isindependently —CH₂CH₃ or —CH₃. In some such embodiments, each Z is —CH₃.

In some embodiments, the chelating agent chelated to the metal atom is:

In some embodiments, the radioactive phospholipid metal chelate compoundis one of the following compounds, wherein the selected compound ischelated to the metal atom:

In some embodiments, the radioactive phospholipid chelate compound isadministered intravenously.

In some embodiments, the subject is a human.

In some embodiments, the method optionally further includes exposing oneof the malignant solid tumors to xRT.

In some embodiments, the method optionally includes the step ofdetermining the immunostimulatory dose of the radioactive phospholipidchelate compound. In some such embodiments, the step of determining theimmunomodulatory dose of the radioactive phospholipid chelate compoundincludes administering to the subject a detection-facilitating dose of aradioactive phospholipid chelate compound as described previously,except that the metal atom is a positron or single photon emitting metalisotope with a half-life of greater than or equal to 4 hours, andsubsequently detecting signals originating from the one or moremalignant solid tumors within the subject that are characteristic of themetal isotope within the radioactive phospholipid chelate compound. Insome such embodiments, the positron or single photon emitting metalisotope is Ga-66, Cu-64, Y-86, Co-55, Zr-89, Sr-83, Mn-52, As-72, Sc-44,Ga-67, In-111, or Tc-99m.

In some embodiments, the immunomodulatory dose of the radioactivephospholipid chelate compound is calculated from the strength of thesignals originating from the one or more malignant solid tumors withinthe subject.

In some embodiments, the step of detecting signals characteristic of themetal isotope is performed by positron emission tomography (PET) imagingor single-photon emission computed tomography (SPECT) imaging.

Non-limiting examples of the cancers presenting as malignant solidtumors that could treated using the disclosed method include melanoma,neuroblastoma, lung cancer, adrenal cancer, colon cancer, colorectalcancer, ovarian cancer, prostate cancer, liver cancer, subcutaneouscancer, squamous cell cancer of the skin or head and neck, intestinalcancer, retinoblastoma, cervical cancer, glioma, breast cancer,pancreatic cancer, soft tissue sarcomas, Ewings sarcoma,rhabdomyosarcoma, osteosarcoma, retinoblastoma, Wilms' tumor, orpediatric brain tumors.

In a second aspect, the disclosure encompasses a method of treating acancer comprising one or more malignant solid tumors in a subject. Themethod includes the steps of systemically administering to the subject(a) an immunomodulatory dose of a targeted radiotherapy (TRT) agent thatis differentially taken up by and retained within the malignant solidtumor tissue; and (b) one or more immunostimulatory agents.

In some embodiments, the one or more immunostimulatory agents are immunecheckpoint inhibitors capable of targeting one or more checkpointmolecules.

Non-limiting examples of the one or more immune checkpoint inhibitorsinclude agents that are capable of targeting one or more of thefollowing checkpoint molecules: A2AR (adenosine A2a receptor), BTLA (Band T lymphocyte attenuator), CTLA4 (cytotoxic T lymphocyte-associatedprotein 4), KIR (killer cell immunoglobulin-like receptor), LAG3(Lymphocyte Activation Gene 3), PD-1 (programmed death receptor 1),PD-L1 (programmed death ligand 1), CD40 (cluster of differentiation 40),CD27 (cluster of differentiation 27), CD28 (cluster of differentiation28), CD137 (cluster of differentiation 137), OX40 (CD134; cluster ofdifferentiation 134), OX40L (OX40 ligand; cluster of differentiation252), GITR (glucocorticoid-induced tumor necrosis factorreceptor-related protein), GITRL (glucocorticoid-induced tumor necrosisfactor receptor-related protein ligand), ICOS (inducible T-cellcostimulatory), ICOSL (inducible T-cell costimulatory ligand), B7H₃(CD276; cluster of differentiation 276), B7H₄ (VTCN1; V-setdomain-containing T-cell activation inhibitor 1), IDO (Indoleamine2,3-dioxygenase), TIM-3 (T-cell Immunoglobulin domain and Mucin domain3), Gal-9 (galectin-9), or VISTA (V-domain Ig suppressor of T cellactivation).

In some embodiments, the one or more immune checkpoint inhibitorsinclude one or more anti-immune checkpoint molecule antibodies. In somesuch embodiments, the one or more anti-immune checkpoint moleculeantibodies include at least one monoclonal antibody.

In some embodiments, the one or more immune checkpoint inhibitorsinclude one or more small molecules capable of inhibiting or blockingone or more immune checkpoint molecules. Non-limiting examples of suchsmall molecule checkpoint inhibitors include CA-170 and CA-327, whichboth target PD-L1.

In some embodiments, the one or more anti-immune checkpoint moleculeantibodies include an anti-CTLA4 antibody, an anti-PD-1 antibody, ananti-PD-L1 antibody, an anti-LAG3 antibody, an anti-KIR antibody, ananti-A2AR antibody, and anti-BTLA antibody, an anti-CD40 antibody, ananti-CD27 antibody, an anti-CD28 antibody, an anti-CD137 antibody, ananti-OX40 antibody, an anti-OX40L antibody, an anti-GITR antibody, ananti-GITRL antibody, an anti-ICOS antibody, an anti-ICOSL antibody, ananti-B7H₃ antibody, an anti-B7H₄ antibody, an anti-IDO antibody, ananti-TIM-3 antibody, an anti-Gal-9 antibody, or an anti-VISTA antibody.

In some embodiments, the TRT agent is metaiodobenzylguanidine (MIBG),where the iodine atom in the MIBG is a radioactive iodine isotope.

In some embodiments, the TRT agent is a radiolabeled tumor-targetingantibody.

In some embodiments, the TRT agent is radioactive isotope of radium,such as Ra-223.

In some embodiments, the TRT agent is a radioactive phospholipid ethermetal chelate having the formula:

or a salt thereof. R₁ includes a chelating agent that is chelated to ametal atom, wherein the metal atom is an alpha, beta or Auger emittingmetal isotope with a half-life of greater than 6 hours and less than 30days; a is 0 or 1; n is an integer from 12 to 30; m is 0 or 1; Y is —H,—OH, —COOH, —COOX, —OCOX, or —OX, wherein X is an alkyl or an aryl; R₂is —N⁺H₃, —N⁺H₂Z, —N⁺HZ₂, or —N⁺Z₃, wherein each Z is independently analkyl or an aroalkyl; and b is 1 or 2. Non-limiting examples of metalisotopes that could be used include Sc-47, Lu-177, Y-90, Ho-166, Re-186,Re-188, Cu-67, Au-199, Rh-105, Ra-223, Ac-225, Pb-212, or Th-227.

In some embodiments, the chelating agent is1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3A) or one of itsderivatives; 1,4,7-triazacyclononane-1,4-diacetic acid (NODA) or one ofits derivatives; 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) orone of its derivatives;1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) or oneof its derivatives; 1,4,7-triazacyclononane,1-glutaric acid-4,7-diaceticacid (NODAGA) or one of its derivatives;1,4,7,10-tetraazacyclodecane,1-glutaric acid-4,7,10-triacetic acid(DOTAGA) or one of its derivatives;1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA) orone of its derivatives;1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4,11-diacetic acid (CB-TE2A)or one of its derivatives; diethylene triamine pentaacetic acid (DTPA),its diester, or one of its derivatives; 2-cyclohexyl diethylene triaminepentaacetic acid (CHX-A″-DTPA) or one of its derivatives; deforoxamine(DFO) or one of its derivatives;1,2-[[6-carboxypyridin-2-yl]methylamino]ethane (H₂dedpa) or one of itsderivatives; and DADA or one of its derivatives, wherein DADA comprisesthe structure:

In some embodiments, a is 1 (aliphatic aryl-alkyl chain). In otherembodiments, a is 0 (aliphatic alkyl chain).

In some embodiments, m is 1 (acylphospholipid series). In some suchembodiments, n is an integer between 12 and 20. In some embodiments, Yis —OCOX, —COOX or —OX.

In some embodiments, X is —CH₂CH₃ or —CH₃.

In some embodiments, m is 0 (alkylphospholipid series).

In some embodiments, b is 1.

In some embodiments, n is 18.

In some embodiments, R₂ is —N⁺Z₃. In some such embodiments, each Z isindependently —CH₂CH₃ or —CH₃. In some such embodiments, each Z is —CH₃.

In some embodiments, the chelating agent chelated to the metal atom is:

In some embodiments, the radioactive phospholipid ether metal chelate isone of the following compounds, wherein the selected compound ischelated to the metal atom:

In some embodiments, in the phospholipid ether metal chelate structure,a is 1, b is 1, m is 0, n is 18, and R₂ is —N⁺(CH₃)₃. In some suchembodiments, the phospholipid ether metal chelate is NM600 chelated tothe metal atom, such as (but not limited to)⁹⁰Y-NM600.

In some embodiments, the TRT agent is a radiohalogenated phospholipidether having the formula:

or a salt thereof. R₁ comprises a radioactive halogen isotope; a is 0 or1; n is an integer from 12 to 30; m is 0 or 1; Y is selected from thegroup consisting of —H, —OH, —COOH, —COOX, —OX, and —OCOX, wherein X isan alkyl or an arylalkyl; and R₂ is selected from the group consistingof —N⁺H₃, —N⁺H₂Z, —N⁺HZ₂, and —N⁺Z₃, wherein each Z is independently analkyl or an aryl.

In some embodiments, the radioactive halogen isotope is ¹²³J, ¹²⁴J,¹²⁵J, ¹³¹J, ²¹¹At, ⁷⁶Br, or ⁷⁷Br.

In some embodiments, a is 1 and m is 0.

In some embodiments, n is 18.

In some embodiments, R₂ is —N⁺(CH₃)3. In some such embodiments, a is 1,m is 0, and n is 18. In some such embodiments, the radioactive halogenisotope is ¹²³I, ¹²⁴I, ¹²⁵I, or ¹³¹I (the radiohalogenated phospholipidether is [¹²³I]—NM404, [¹²⁴I]—NM404, [¹²5I]—NM404, [¹³¹I]—NM404,[²¹¹At]—NM404, [⁷⁶Br]—NM404, or [⁷⁷Br]—NM404).

In some embodiments, the TRT agent, the immunostimulatory agent, orboth, are administered intravenously.

In some embodiments, the subject is a human.

Non-limiting examples of the cancers presenting as malignant solidtumors that can be treated using the method include melanoma,neuroblastoma, lung cancer, adrenal cancer, colon cancer, colorectalcancer, ovarian cancer, prostate cancer, liver cancer, subcutaneouscancer, squamous cell cancer of the skin or head or neck, intestinalcancer, retinoblastoma, cervical cancer, glioma, breast cancer,pancreatic cancer, soft tissue sarcoma, Ewings sarcoma,rhabdomyosarcoma, osteosarcoma, Wilms' tumor, and pediatric braintumors.

In some embodiments, the cancer is treated without administering to thesubject an antibody to a tumor antigen that is not a checkpointmolecule.

In some embodiments, an anti-GD2 antibody is not adminstered to thesubject.

Other objects, features and advantages of the present invention willbecome apparent after review of the specification, claims and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 shows the chemical structure of the base compound18-(p-iodophenyl) octadecyl phosphocholine (NM404).

FIG. 2A is a graph showing that xRT+IT-IC elicits in situ tumorvaccination. More specifically, FIG. 2A shows tumor growth curves thatshow synergy between xRT and IT-hu14.18-IL2. 71% ( 22/31) of micetreated with xRT+IT-IC are rendered disease-free.

FIG. 2B is another graph showing that xRT+IT-IC elicits in situ tumorvaccination. More specifically, FIG. 2B shows Kaplan-Meier survivalcurves that show synergy between xRT and IT-hu14.18-IL2. 71% ( 22/31) ofmice treated with xRT+IT-IC are rendered disease-free.

FIG. 2C is another graph showing that xRT+IT-IC elicits in situ tumorvaccination. More specifically, FIG. 2C shows that 90% of the treatedmice reject subsequent engraftment with B78 melanoma.

FIG. 3 is a graph demonstrating concomitant immune tolerance. Primarytumor response is shown. A distant un-treated tumor suppresses responseto xRT+IT-IC in a 2-tumor B78 melanoma model, and this suppression canbe overcome be radiating the second tumor.

FIG. 4 is a graph showing that concomitant immune tolerance is due toTregs. Primary tumor response is shown. A distant un-treated tumorsuppresses response to xRT+IT-IC in a 2-tumor B78 melanoma model andthis suppression can be overcome by depleting Tregs (using transgenicDEREG mice that express diphtheria toxin receptors on their Tregs, andthus depleting Tregs by administering diphtheria toxin).

FIG. 5 is an image showing selective uptake of ¹²⁴I-NM404 by B78melanoma. A mouse bearing a ˜200 mm³ B78 tumor received IV ¹²⁴I-NM404and had serial PET/CT scans done. This image at 71 h shows selectiveuptake by the tumor with some residual background uptake by the heartand liver.

FIG. 6 is a graph demonstrating that in situ vaccination can be elicitedin the presence of residual levels of molecular targeted radiationtherapy (TRT). Treatment with combined xRT+IT-IC is equally effective inthe presence or absence of 3 μCi ¹³¹I-NM404. This approximates theresidual activity of TRT that will be present when we deliver xRT (d0)followed by IT-IC (d6-10), as described in Example 4.

FIG. 7 shows a time course MRI image of a tumor-bearing mouse followinginjection of Gd-NM600 showing enhancement of the tumor (T) by 24 hours.

FIG. 8A is a graph showing tumor-specific inhibition of primary tumorresponse to the combination of local RT+IT-IC by a distant untreatedtumor in murine melanoma and pancreatic tumor models. C57BL/6 micebearing a syngeneic, disialoganglioside-expressing (GD2+), primary flanktumor +/−a secondary tumor on the contralateral flank were treated tothe primary tumor only, as indicated, with xRT on day “1” andintra-tumor (IT) injection of 50 mcg of the anti-GD2 immunocytokine(IC), hu14.18-IL2 (a fusion of anti-GD2 mAb and IL2), on day 6-10. Meanprimary tumor volumes are displayed in FIG. 8A. More specifically, FIG.8A shows that in mice bearing a primary B78 melanoma tumor, the presenceof an untreated secondary B78 tumor antagonized primary tumor responseto RT+IT-IC. We describe this effect as “concomitant immunetolerance”—an antagonistic effect of a non-treated distant tumor on thelocal response of a treated tumor to xRT+IT-IC.

FIG. 8B is another graph showing tumor-specific inhibition of primarytumor response to the combination of local RT+IT-IC by a distantuntreated tumor in murine melanoma and pancreatic tumor models. C57BL/6mice bearing a syngeneic, disialoganglioside-expressing (GD2+), primaryflank tumor +/−a secondary tumor on the contralateral flank were treatedto the primary tumor only, as indicated, with xRT on day “1” andintra-tumor (IT) injection of 50 mcg of the anti-GD2 immunocytokine(IC), hu14.18-IL2 (a fusion of anti-GD2 mAb and IL2), on day 6-10. Morespecifically, FIG. 8B shows Kaplan-Meier survival curves for mice plusreplicate experiments. Nearly all mice were euthanized due to primarytumor progression.

FIG. 8C is another graph showing tumor-specific inhibition of primarytumor response to the combination of local RT+IT-IC by a distantuntreated tumor in murine melanoma and pancreatic tumor models. C57BL/6mice bearing a syngeneic, disialoganglioside-expressing (GD2+), primaryflank tumor +/−a secondary tumor on the contralateral flank were treatedto the primary tumor only, as indicated, with xRT on day “1” andintra-tumor (IT) injection of 50 mcg of the anti-GD2 immunocytokine(IC), hu14.18-IL2 (a fusion of anti-GD2 mAb and IL2), on day 6-10. Morespecifically, FIG. 8C shows that in mice bearing a primary Panc02-GD2+pancreatic tumor, with or without a secondary Panc02-GD2-tumor on theopposite flank, the presence of an untreated Panc02 secondary tumorsuppressed the response of a primary Panc02-GD2+ tumor to RT+IT-IC.

FIG. 8D is another graph showing tumor-specific inhibition of primarytumor response to the combination of local RT+IT-IC by a distantuntreated tumor in murine melanoma and pancreatic tumor models. C57BL/6mice bearing a syngeneic, disialoganglioside-expressing (GD2+), primaryflank tumor +/−a secondary tumor on the contralateral flank were treatedto the primary tumor only, as indicated, with xRT on day “1” andintra-tumor (IT) injection of 50 mcg of the anti-GD2 immunocytokine(IC), hu14.18-IL2 (a fusion of anti-GD2 mAb and IL2), on day 6-10. Morespecifically, FIG. 8D shows that in mice bearing a primary B78 melanomatumor, a secondary B78 tumor suppressed primary tumor response toRT+IT-IC but a secondary Panc02-GD2+ pancreatic tumor did not exert thiseffect.

FIG. 8E is another graph showing tumor-specific inhibition of primarytumor response to the combination of local RT+IT-IC by a distantuntreated tumor in murine melanoma and pancreatic tumor models. C57BL/6mice bearing a syngeneic, disialoganglioside-expressing (GD2+), primaryflank tumor +/−a secondary tumor on the contralateral flank were treatedto the primary tumor only, as indicated, with xRT on day “1” andintra-tumor (IT) injection of 50 mcg of the anti-GD2 immunocytokine(IC), hu14.18-IL2 (a fusion of anti-GD2 mAb and IL2), on day 6-10. Morespecifically, FIG. 8E shows that in mice bearing a primary Panc02-GD2+tumor a secondary Panc02-GD2-tumor suppressed primary tumor response tocombined xRT and IT-hu14.18-IL2, while a B78 secondary tumor did not.n=number of mice per group. NS=non-significant, ***p<0.001.

FIG. 9A includes immunohistochemistry images (left and center) andgraphs (right) showing that concomitant immune tolerance is circumventedby specific depletion of regulator T cells (Tregs). More specifically,FIG. 9A shows immunohistochemistry for the Treg marker, FoxP3(representative 400× images are shown) for tumors evaluated on day 6after xRT in mice with one (FIG. 9A, leftmost panels A1 and A2) or two(FIG. 9A center panels A3 and A4) tumors. Mice received no xRT, or xRTonly to the primary tumor. The primary tumor is shown in FIG. 9A,panels_A1-A3 and the secondary is shown in FIG. 9A, panel A4. Smallarrows point out some of the FoxP3+ cells (brown nuclei=FoxP3+,blue=hematoxylin counterstain). The graphs on the right display blindedquantification of FoxP3+ cells per 200× field, corresponding to theconditions shown in FIG. 9A, panels A1, A2, A3 and A4, respectively.

FIG. 9B is another graph showing that concomitant immune tolerance iscircumvented by specific depletion of regulator T cells (Tregs). Morespecifically, FIG. 9B shows that DEREG mice express diphtheria toxinreceptor under control of the Treg-specific FoxP3 promoter, enablingspecific depletion of Tregs upon IP injection of diphtheria toxin. DEREGmice bearing primary and secondary B78 melanoma tumors were treated withxRT+IT-IC to the primary tumor and IP injection of either diphtheriatoxin or PBS (the first of replicate experiments are shown). Concomitantimmune tolerance is eliminated following depletion of Tregs in thesemice, resulting in improved (FIG. 9B) primary tumor response. n=numberof mice per group. **p<0.01, ***p<0.001.

FIG. 9C is another graph showing that concomitant immune tolerance iscircumvented by specific depletion of regulator T cells (Tregs). Morespecifically, FIG. 9C shows that DEREG mice express diphtheria toxinreceptor under control of the Treg-specific FoxP3 promoter, enablingspecific depletion of Tregs upon IP injection of diphtheria toxin. DEREGmice bearing primary and secondary B78 melanoma tumors were treated withxRT+IT-IC to the primary tumor and IP injection of either diphtheriatoxin or PBS (the first of replicate experiments are shown). Concomitantimmune tolerance is eliminated following depletion of Tregs in thesemice, resulting in improved (FIG. 9C) secondary tumor response. n=numberof mice per group. **p<0.01, ***p<0.001.

FIG. 10A and is a graph showing that concomitant immune tolerance isovercome by delivering xRT to both tumor sites. In mice bearing primaryand secondary B78 tumors, the secondary tumor suppresses primary tumorresponse to primary tumor treatment with xRT+IT-IC. This is overcome bydelivering 12 Gy xRT to both the primary and secondary tumors and IT-ICto the primary tumor, resulting in improved (FIG. 10A) primary tumorresponse (the first of replicate experiments is shown) from replicateexperiments. n=number of mice per group. **p<0.01, ***p<0.001.

FIG. 10B is another graph showing that concomitant immune tolerance isovercome by delivering xRT to both tumor sites. In mice bearing primaryand secondary B78 tumors, the secondary tumor suppresses primary tumorresponse to primary tumor treatment with xRT+IT-IC. This is overcome bydelivering 12 Gy xRT to both the primary and secondary tumors and IT-ICto the primary tumor, resulting in improved (FIG. 10B) aggregate animalsurvival from replicate experiments. n=number of mice per group.**p<0.01, ***p<0.001.

FIG. 11A is a graph showing that low dose xRT alone does not elicit insitu vaccination but does overcome concomitant immune tolerance whendelivered to distant tumor sites together with 12 Gy+IT-IC treatment ofan in situ vaccine site. More specifically, FIG. 11A shows that in micebearing a primary B78 tumor only, 12 Gy+IT-IC elicits in situvaccination (as shown previously) and results in complete tumorregression in most mice ( 4/6 in this experiment) and a memory immuneresponse (Morris, Cancer Res, 2016). On the other hand no animalsexhibit complete tumor regression following either IT-IC alone or lowdose (2 Gy) xRT+IT-IC ( 0/6 in both groups) p<0.05.

FIG. 11B is another graph showing that low dose xRT alone does notelicit in situ vaccination but does overcome concomitant immunetolerance when delivered to distant tumor sites together with 12Gy+IT-IC treatment of an in situ vaccine site. More specifically, FIG.11B shows that in mice bearing a primary and secondary B78 melanomatumor, low dose xRT (2 Gy or 5 Gy) delivered to the secondary tumor iscomparable to 12 Gy in its capacity to overcome concomitant immunetolerance at the primary tumor.

FIG. 11C is another graph showing that low dose xRT alone does notelicit in situ vaccination but does overcome concomitant immunetolerance when delivered to distant tumor sites together with 12Gy+IT-IC treatment of an in situ vaccine site. More specifically, FIG.11C shows that in these same animals, it is apparent that overcomingconcomitant immune tolerance by delivery of low dose xRT to thesecondary tumor rescues a systemic response to IT-IC immunotherapy. Inthis context, when xRT is delivered to all tumor sites then IT-ICinjection of the primary tumor triggers a systemic anti-tumor effectthat renders secondary tumor response to 2 Gy or 5 Gy greater than theresponse to 12 Gy xRT in absence of primary tumor IT-IC injection.

FIG. 12A is a PET image showing that low dose TRT with ¹³¹I-NM404effectively depletes tumor infiltrating FoxP3+ Tregs without systemicleukopenia or depletion of tumor infiltrating CD8+ effector T cells. Inmost clinical scenarios, it is not feasible to deliver external beam,even low dose, to all tumor sites without eliciting marked bone marrowdepletion and leukopenia that would result in immunosuppression. Here wetested whether TRT could be administered systemically to specificallydeplete tumor infiltrating suppressive immune cells (Tregs), withouttriggering systemic immune cell depletion and leukopenia. Morespecifically, FIG. 12A shows dosimetry studies in this B78 melanomatumor model using positron-emitting ¹²⁴I-NM404 confirm tumor-selectiveuptake of NM404. C57BL/6 mice bearing B78 tumors were treated with 60μCi ¹³¹I-NM404. This activity approximates the amount of 131I-NM404necessary to deliver 2 Gy TRT to a B78 tumor. Peripheral blood and tumorsamples were collected in untreated control mice (designated “C”) and at8 day intervals (T1=d8, T2=d16, T3=d24, T4=d32) thereafter.

FIG. 12B is a bar graph showing that low dose TRT with ¹³¹I-NM404effectively depletes tumor infiltrating FoxP3+ Tregs without systemicleukopenia or depletion of tumor infiltrating CD8+ effector T cells. Inmost clinical scenarios, it is not feasible to deliver external beam,even low dose, to all tumor sites without eliciting marked bone marrowdepletion and leukopenia that would result in immunosuppression. Here wetested whether TRT could be administered systemically to specificallydeplete tumor infiltrating suppressive immune cells (Tregs), withouttriggering systemic immune cell depletion and leukopenia. Morespecifically, FIG. 12B shows that this dose of TRT did not result in anysignificant systemic leukopenia.

FIG. 12C is another bar graph showing that low dose TRT with ¹³¹I-NM404effectively depletes tumor infiltrating FoxP3+ Tregs without systemicleukopenia or depletion of tumor infiltrating CD8+ effector T cells. Inmost clinical scenarios, it is not feasible to deliver external beam,even low dose, to all tumor sites without eliciting marked bone marrowdepletion and leukopenia that would result in immunosuppression. Here wetested whether TRT could be administered systemically to specificallydeplete tumor infiltrating suppressive immune cells (Tregs), withouttriggering systemic immune cell depletion and leukopenia. Morespecifically, FIG. 12C shows that this dose of TRT did not significantlyaffect the level of tumor infiltrating CD8+ effector T cells (ANOVAp=0.25).

FIG. 12D is another bar graph showing that low dose TRT with ¹³¹I-NM404effectively depletes tumor infiltrating FoxP3+ Tregs without systemicleukopenia or depletion of tumor infiltrating CD8+ effector T cells. Inmost clinical scenarios, it is not feasible to deliver external beam,even low dose, to all tumor sites without eliciting marked bone marrowdepletion and leukopenia that would result in immunosuppression. Here wetested whether TRT could be administered systemically to specificallydeplete tumor infiltrating suppressive immune cells (Tregs), withouttriggering systemic immune cell depletion and leukopenia. Morespecifically, FIG. 12D shows that tumor infiltrating FoxP3+ Tregs weresignificantly depleted by this dose of TRT (ANOVA p=0.03; * p<0.05).

FIG. 13A is a graph showing that low dose TRT with ¹³¹I-NM404effectively overcomes concomitant immune tolerance and rescues thesystemic anti-tumor effect of in situ vaccination. Given the capacity oflow dose ¹³¹I-NM404 TRT to deplete tumor-infiltrating Tregs withoutrendering a mouse leukopenic, we tested whether low dose ¹³¹I-NM404might effectively overcome concomitant immune tolerance. C57BL/6 micebearing two B78 tumors were treated with 60-mcCi ¹³¹I-NM404 on day 1(NM404), as indicated. After one half-life (day 8), animals received 12Gy xRT or no xRT to the primary tumor (in situ vaccine site). Controlmice receiving no ¹³¹I-NM404 were treated to the secondary tumor asindicated (0, 2, or 12 Gy). Mice received daily IT injections of IC tothe primary tumor (in situ vaccine site), as indicated, on days 13-17.More specifically, FIG. 13A shows that primary tumor response is shownand demonstrates that administration of low dose TRT effectivelyovercomes concomitant immune tolerance and rescues the systemicanti-tumor effect of in situ vaccination.

FIG. 13B is another graph showing that low dose TRT with ¹³¹I-NM404effectively overcomes concomitant immune tolerance and rescues thesystemic anti-tumor effect of in situ vaccination. Given the capacity oflow dose ¹³¹I-NM404 TRT to deplete tumor-infiltrating Tregs withoutrendering a mouse leukopenic, we tested whether low dose ¹³¹I-NM404might effectively overcome concomitant immune tolerance. C57BL/6 micebearing two B78 tumors were treated with 60-mcCi ¹³¹I-NM404 on day 1(NM404), as indicated. After one half-life (day 8), animals received 12Gy xRT or no xRT to the primary tumor (in situ vaccine site). Controlmice receiving no ¹³¹I-NM404 were treated to the secondary tumor asindicated (0, 2, or 12 Gy). Mice received daily IT injections of IC tothe primary tumor (in situ vaccine site), as indicated, on days 13-17.More specifically, FIG. 13B shows that secondary tumor response is shownand demonstrates that administration of low dose TRT effectivelyovercomes concomitant immune tolerance and rescues the systemicanti-tumor effect of in situ vaccination.

FIG. 14 shows the chemical structure of an exemplary alkylphosphocholinemetal chelate (⁶⁴Cu-NM600). Other metals may be used in place of ⁶⁴Cu.

FIG. 15 is a PET/CT image of two single tumor B78 mice from a scan taken48 hours post-injection with ⁸⁶Y-NM600.

FIG. 16 is a PET/CT image of two two-tumor B78 mice from a scan taken 48hours post-injection with ⁸⁶Y-NM600.

FIG. 17 includes PET/CT images for a U87MG mouse from scans taken 3hours (left panel), 24 hours (center panel) and 48 hours (right panel)post-injection with ⁶⁴Cu-NM600. The images show tissue activitycalculated as a percent of injected dose/g tissue (% ID/g, scale shownon far right).

FIG. 18 includes PET/CT images for a 4T1 mouse from scans taken 3 hours(left panel), 24 hours (center panel) and 48 hours (right panel)post-injection with ⁶⁴Cu-NM600. The images show tissue activitycalculated as a percent of injected dose/g tissue (% ID/g, scale shownon far right).

FIG. 19 includes PET/CT images for an HCT-116 mouse from scans taken 3hours (left panel), 24 hours (center panel) and 48 hours (right panel)post-injection with ⁶⁴Cu-NM600. The images show tissue activitycalculated as a percent of injected dose/g tissue (% ID/g, scale shownon far right).

FIG. 20 includes PET/CT images for an A549 mouse from scans taken 3hours (left panel), 24 hours (center panel) and 48 hours (right panel)post-injection with ⁶⁴Cu-NM600. The images show tissue activitycalculated as a percent of injected dose/g tissue (% ID/g, scale shownon far right).

FIG. 21 includes PET/CT images for a PC-3 mouse from scans taken 3 hours(left panel), 24 hours (center panel) and 48 hours (right panel)post-injection with ⁶⁴Cu-NM600. The images show tissue activitycalculated as a percent of injected dose/g tissue (% ID/g, scale shownon far right).

FIG. 22 includes PET/CT images for an HT-29 mouse from scans taken 3hours (left panel), 24 hours (center panel) and 48 hours (right panel)post-injection with ⁶⁴Cu-NM600. The images show tissue activitycalculated as a percent of injected dose/g tissue (% ID/g, scale shownon far right).

FIG. 23 includes PET/CT images for a MiaPaca mouse from scans taken 3hours (left panel), 24 hours (center panel) and 48 hours (right panel)post-injection with ⁶⁴Cu-NM600. The images show tissue activitycalculated as a percent of injected dose/g tissue (% ID/g, scale shownon far right).

FIG. 24 includes PET/CT images for a 4T1 mouse from scans taken 3 hours(left panel), 24 hours (center panel) and 48 hours (right panel)post-injection with ⁸⁶Y-NM600. The images show tissue activitycalculated as a percent of injected dose/g tissue (% ID/g, scale shownon far right).

FIG. 25 includes PET/CT images for a 4T1 mouse from scans taken 3 hours(left panel), 24 hours (center panel) and 48 hours (right panel)post-injection with ⁸⁹Zr-NM600. The images show tissue activitycalculated as a percent of injected dose/g tissue (% ID/g, scale shownon far right).

FIG. 26 includes PET/CT images for an HT-29 mouse from scans taken 4hours (left panel) and 1 day (right panel) post-injection with⁵²Mn-NM600. The images show tissue activity calculated as a percent ofinjected dose/g tissue (% ID/g, scale shown on far right).

FIG. 27 includes PET/CT images for a PC-3 mouse from scans taken 4 hours(left panel) and 1 day (right panel) post-injection with ⁵²Mn-NM600. Theimages show tissue activity calculated as a percent of injected dose/gtissue (% ID/g, scale shown to the right of each image).

FIG. 28 includes PET/CT images for an HT-29 mouse from scans taken 2days (left panel), 3 days (second panel from the left), 5 days (secondpanel form the right) and 7 days (right panel) post-injection with⁵²Mn-NM600. The images show tissue activity calculated as a percent ofinjected dose/g tissue (% ID/g, scale shown to the right of the images).

FIG. 29 includes PET/CT images for a PC-3 mouse from scans taken 2 days(left panel), 3 days (second panel from the left), 5 days (second panelform the right) and 7 days (right panel) post-injection with ⁵²Mn-NM600.The images show tissue activity calculated as a percent of injecteddose/g tissue (% ID/g, scale shown to the right of the images).

FIG. 30 is a graph showing PET quantitative region of interest data(chelate uptake as a function of time) for 4T1 tumor tissue in 4T1 miceinjected with ⁸⁶Y-NM600, ⁶⁴Cu-NM600 and ⁸⁹Zr-NM-600.

FIG. 31 is a graph showing PET quantitative region of interest data(chelate uptake as a function of time) for heart tissue in 4T1 miceinjected with ⁸⁶Y-NM600, ⁶⁴Cu-NM600 and ⁸⁹Zr-NM-600.

FIG. 32 is a graph showing PET quantitative region of interest data(chelate uptake as a function of time) for liver tissue in 4T1 miceinjected with ⁸⁶Y-NM600, ⁶⁴Cu-NM600 and ⁸⁹Zr-NM-600.

FIG. 33 is a graph showing PET quantitative region of interest data(chelate uptake as a function of time) for whole body in 4T1 miceinjected with ⁸⁶Y-NM600, ⁶⁴Cu-NM600 and ⁸⁹Zr-NM-600.

FIG. 34 is a bar graph illustrating ex vivo chelate biodistribution inhealthy and tumor tissues in 4T1 mice 48 hours (⁸⁶Y-NM600, ⁶⁴Cu-NM600,⁸⁹Zr-NM-600 and ¹⁷⁷Lu-NM600) and 96 hours (¹⁷⁷Lu-NM600) post-injectionof the metal chelates.

FIG. 35 shows the chemical structure of an exemplary alkylphosphocholinemetal chelate (¹⁷⁷Lu-NM600). Other metals may be used in place of ¹⁷⁷Lu.

FIG. 36 is an audioradiographic image of three B78 mice taken 48 hoursafter injection with ⁹⁰Y-NM600. Xenografted B78 tumors are seen as largedark spots at the lower right of each mouse image.

FIG. 37 is an audioradiographic image of three B78 mice taken 96 hoursafter injection with ⁹⁰Y-NM600. Xenografted B78 tumors are seen as largedark spots at the lower right of each mouse image.

FIG. 38 is an audioradiographic image of a B78 mouse taken on day 5after injection with ¹⁷⁷Lu-NM600. Xenografted B78 tumors are seen as twodark spots at the bottom of the mouse image.

FIG. 39 is an audioradiographic image of a B78 mouse taken on day 13after injection with ¹⁷⁷Lu-NM600. Xenografted B78 tumors are seen as twodark spots at the bottom of the mouse image.

FIG. 40 is an audioradiographic image of a MiaPaca mouse taken 10 daysafter injection with ¹⁷⁷Lu-NM600. The location of the xenograftedMiaPaca tumor is indicated by the arrow and dashed circle.

FIG. 41 is an audioradiographic image of three 4T1 mice taken 48 hoursafter injection with ¹⁷⁷Lu-NM600. The locations of the xenografted 4T1tumors are indicated by the arrows and dashed circles.

FIG. 42 is an audioradiographic image of three 4T1 mice taken 96 hoursafter injection with ¹⁷⁷Lu-NM600. The locations of the xenografted 4T1tumors are indicated by the dashed circles.

FIG. 43 is an audioradiographic image of three 4T1 mice taken 4 hoursafter injection with ⁹⁰Y-NM600. The locations of the xenografted 4T1tumors are indicated by the arrows and dashed circles.

FIG. 44 is an audioradiographic image of three 4T1 mice taken 48 hoursafter injection with ⁹⁰Y-NM600. The xenografted 4T1 tumors are seen aslarge dark spots on the lower right of each mouse image.

FIG. 45 is an audioradiographic image of three 4T1 mice taken 96 hoursafter injection with ⁹⁰Y-NM600. The xenografted 4T1 tumors are seen aslarge dark spots on the lower right of each mouse image.

FIG. 46 is a graph illustrating the radiotherapeutic effect of ⁹⁰Y-NM600at two different doses (150 μCi and 300 μCi) in a B78 xenograft mousemodel, versus a control (excipient only). Data is presented as measuredtumor volume in mm³ as a function of time in days after injection.

FIG. 47 is a graph illustrating the radiotherapeutic effect of a single500 μCi dose of ¹⁷⁷Lu-NM600 in a B78 xenograft mouse model, versus acontrol (excipient only). Data is presented as measured tumor volume inmm³ as a function of time in days after injection.

FIG. 48 is a graph illustrating the radiotherapeutic effect of a single400 μCi dose of ¹⁷⁷Lu-NM600 in a MiaPaca xenograft mouse model, versus acontrol (excipient only). Data is presented as measured tumor volume inmm³ as a function of time in days after injection.

FIG. 49 is a graph illustrating the radiotherapeutic effect of a single500 μCi dose of ¹⁷⁷Lu-NM600 in a 4T1 xenograft mouse model, versus acontrol (excipient only). Data is presented as measured tumor volume inmm³ as a function of time in days after injection. *P<0.05; **P<0.01;***P<0.001.

FIG. 50 is a graph illustrating the radiotherapeutic effect of twoserial doses of ¹⁷⁷Lu-NM600 (500 μCi and 250 μCi) in a 4T1 xenograftmouse model, versus a control (excipient only). Data is presented asmeasured tumor volume in mm³ as a function of time in days afterinjection.

FIG. 51 is a graph illustrating the radiotherapeutic effect of¹⁷⁷Lu-NM600 at two different doses (500 μCi and 250 μCi) in a 4T1xenograft mouse model, versus a control (excipient only). Data ispresented as measured tumor volume in mm³ as a function of time in daysafter injection.

FIG. 52 is a graph illustrating the impact of tumor mass on thecomparative therapeutic efficacy of ⁹⁰Y-NM600 and ¹³¹I-NM404 inconventional TRT.

FIG. 53 is a bar graph comparing average albumin binding energies ofthree different metal chelate analogs of NM404, along with an amineanalog. For comparison, the binding energy of I-NM404 is shown as adotted line.

FIG. 54 is a graph illustrating tumor volume (mm³) as a function of time(days) in B78 melanoma flank tumor mice treated with anti-CTLA4 immunecheckpoint inhibitor (CTLA4) and/or varying doses (25 μCi, 50 μCi, or100 μCi) of the targeted radiotherapy (TRT) agent Y90-NM600. Controlmice were administered vehicle without anti-CTLA4 or the TRT agent(PBS). After Day 18, combination treatment with 50 or 100 μCi ofY90-NM600 with anti-CTLA4 had significantly (p<0.05 by ANOVA) reducedtumor growth compared to PBS, Y90-NM600 alone, or anti-CTLA4 alone. The25 μCi Y90-NM600 combination treatment group with anti-CTLA-4 had anintermediate growth delay response that showed a trend towards doseresponse.

FIG. 55 is a graph showing aggregate animal survival for miceadministered a combination of TRT (50 μCi Y90-NM600) and checkpointblockade (anti-CTLA4), compared to mice administered TRT alone,checkpoint blockade alone (anti-CTLA4), or PBS vehicle.

FIG. 56 is a graph showing aggregate animal survival for miceadministered three different combinations of TRT (25 μCi, 50 μCi, and100 μCi Y90-NM600) with checkpoint blockade (anti-CTLA4).

DETAILED DESCRIPTION I. In General

It is understood that this disclosure is not limited to the particularmethodology, protocols, materials, and reagents described, as these mayvary. The terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to limit the scope ofthe present invention, which will be limited only by any later-filednonprovisional applications.

As used herein and in the appended claims, the singular forms “a”, “an”,and “the” include plural reference unless the context clearly dictatesotherwise. As well, the terms “a” (or “an”), “one or more” and “at leastone” can be used interchangeably herein. The terms “comprising” andvariations thereof do not have a limiting meaning where these termsappear in the description and claims. Accordingly, the terms“comprising”, “including”, and “having” can be used interchangeably.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications and patentsspecifically mentioned herein are incorporated by reference for allpurposes including describing and disclosing the chemicals, instruments,statistical analysis and methodologies which are reported in thepublications which might be used in connection with the invention. Allreferences cited in this specification are to be taken as indicative ofthe level of skill in the art.

The terminology as set forth herein is for description of theembodiments only and should not be construed as limiting of theinvention as a whole. Unless otherwise specified, “a,” “an,” “the,” and“at least one” are used interchangeably and mean one or more than one.

The disclosure is inclusive of the compounds described herein (includingintermediates) in any of their pharmaceutically acceptable forms,including isomers (e.g., diastereomers and enantiomers), tautomers,salts, solvates, polymorphs, prodrugs, and the like. In particular, if acompound is optically active, the invention specifically includes eachof the compound's enantiomers as well as racemic mixtures of theenantiomers. It should be understood that the term “compound” includesany or all of such forms, whether explicitly stated or not (although attimes, “salts” are explicitly stated).

“Pharmaceutically acceptable” as used herein means that the compound orcomposition or carrier is suitable for administration to a subject toachieve the treatments described herein, without unduly deleterious sideeffects in light of the necessity of the treatment.

The term “effective amount,” as used herein, refers to the amount of thecompounds or dosages that will elicit the biological or medical responseof a subject, tissue or cell that is being sought by the researcher,veterinarian, medical doctor or other clinician.

As used herein, “pharmaceutically-acceptable carrier” includes any andall dry powder, solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic agents, absorption delaying agents, and thelike. Pharmaceutically-acceptable carriers are materials, useful for thepurpose of administering the compounds in the method of the presentinvention, which are preferably non-toxic, and may be solid, liquid, orgaseous materials, which are otherwise inert and pharmaceuticallyacceptable, and are compatible with the compounds of the presentinvention. Examples of such carriers include, without limitation,various lactose, mannitol, oils such as corn oil, buffers such as PBS,saline, polyethylene glycol, glycerin, polypropylene glycol,dimethylsulfoxide, an amide such as dimethylacetamide, a protein such asalbumin, and a detergent such as Tween 80, mono- andoligopolysaccharides such as glucose, lactose, cyclodextrins and starch.

The term “administering” or “administration,” as used herein, refers toproviding the compound or pharmaceutical composition of the invention toa subject suffering from or at risk of the diseases or conditions to betreated or prevented.

A route of administration in pharmacology is the path by which a drug istaken into the body. Routes of administration may be generallyclassified by the location at which the substance is applied. Commonexamples may include oral and intravenous administration. Routes canalso be classified based on where the target of action is. Action may betopical (local), enteral (system-wide effect, but delivered through thegastrointestinal tract), or parenteral (systemic action, but deliveredby routes other than the GI tract), via lung by inhalation. One form oflocal administration referred to in this submission is intratumoral(IT), whereby an agent is injected directly into, or adjacent to, aknown tumor site.

A topical administration emphasizes local effect, and substance isapplied directly where its action is desired. Sometimes, however, theterm topical may be defined as applied to a localized area of the bodyor to the surface of a body part, without necessarily involving targeteffect of the substance, making the classification rather a variant ofthe classification based on application location. In an enteraladministration, the desired effect is systemic (non-local), substance isgiven via the digestive tract. In a parenteral administration, thedesired effect is systemic, and substance is given by routes other thanthe digestive tract.

Non-limiting examples for topical administrations may includeepicutaneous (application onto the skin), e.g., allergy testing ortypical local anesthesia, inhalational, e.g. asthma medications, enema,e.g., contrast media for imaging of the bowel, eye drops (onto theconjunctiva), e.g., antibiotics for conjunctivitis, ear drops, such asantibiotics and corticosteroids for otitis externa, and those throughmucous membranes in the body.

Enteral administration may be administration that involves any part ofthe gastrointestinal tract and has systemic effects. The examples mayinclude those by mouth (orally), many drugs as tablets, capsules, ordrops, those by gastric feeding tube, duodenal feeding tube, orgastrostomy, many drugs and enteral nutrition, and those rectally,various drugs in suppository.

Examples of parenteral administrations may include intravenous (into avein), e.g. many drugs, total parenteral nutrition intra-arterial (intoan artery), e.g., vasodilator drugs in the treatment of vasospasm andthrombolytic drugs for treatment of embolism, intraosseous infusion(into the bone marrow), intra-muscular, intracerebral (into the brainparenchyma), intracerebroventricular (into cerebral ventricular system),intrathecal (an injection into the spinal canal), and subcutaneous(under the skin). Among them, intraosseous infusion is, in effect, anindirect intravenous access because the bone marrow drains directly intothe venous system. Intraosseous infusion may be occasionally used fordrugs and fluids in emergency medicine and pediatrics when intravenousaccess is difficult.

The following abbreviations are used in this disclosure: ADCC, Antibodydependent cell-mediated cytotoxicity; anti-CTL4, an antibody thattargets cytotoxic T lymphocyte-associated antigen 4 (CTLA4), which isfound on cytotoxic T lymphocytes (CTLs); B16, A melanoma syngeneic toC57B1/6 mice; B78, A variant of B16 that expresses GD2, due totransfection with GD2 synthase; D, day; Hul4.18-IL2, The primaryimmunocytokine (reacts against GD2) used in the studies disclosed in theexamples; IC, Immunocytokine (a fusion protein of a tumor-reactive mAblinked to IL2); ICI, immune checkpoint inhibitor; IL2, Interleukin 2;IT, Intratumoral; IV, Intravenous; mAb, Monoclonal antibody; MAHA, Mouseanti-human antibody; NM404, used to designate the phospholipid ethershown in FIG. 1 , which is selectively taken up by most tumors and usedfor TRT in the studies disclosed in the examples; NM600, used todesignate the phospholipid ether shown in FIG. 14 , which can bechelated with any metal, and which is also selectively taken up by mosttumors and used for TRT in the studies disclosed in the examples; NXS2,A neuroblastoma syngeneic to AJ mice; Panc02-GD2, A pancreatic cancersyngeneic to C57B1/6 mice, expressing GD2, due to transfection with GD2synthase; PLE, Phospholipid ether; RT, Radiation therapy; TRT, Targetedradiotherapy; W, week; 9464D-GD2, A neuroblastoma syngeneic to C57B1/6mice, expressing GD2, due to transfection with GD2 synthase.

II. The Invention

This disclosure is directed to methods of treating any cancer thatpresents as one or malignant solid tumors. The disclosed methods combinetwo treatment steps, with an unexpected synergy resulting in a muchimproved in situ vaccination effect against the malignant solid tumors.Specifically, an immunomodulatory dose of a radioactive phospholipidmetal chelate compound that is differentially taken up by and retainedwithin malignant solid tumor tissue is administered to the patient, andin situ tumor vaccination is performed by intratumorally injecting into(or applying to) at least one of the malignant solid tumors acomposition that includes one or more agents capable of stimulatingspecific immune cells within the tumor microenvironment, either with orwithout additional xRT to at least one of the malignant solid tumorsbeing treated with immune-stimulating agents. The immunomodulatory doseof the radioactive phospholipid metal chelate compound likely reducesTreg levels (and other immune-suppressive elements) and prevents theimmune system dampening (concomitant immune tolerance) that occurs whenxRT is used against a tumor and one or more additional tumors are notradiated, although an understanding of the mechanism is not necessary topractice the invention and the invention is not limited to anyparticular mechanism of action.

A. Intratumoral Immunization—In Situ Vaccination

Intratumoral immunization by in situ vaccination is performed by locallyadministering to the tumor (e.g., by intratumoral injection) one or moreagents capable of stimulating specific immune cells within the tumormicroenvironment. Compositions used for intratumoral immunization mayinclude, without limitation, one or more cytokines, immune checkpointinhibitors, pattern recognition agonists, and/or immunostimulatorymonoclonal antibodies, including antibodies against tumor-specificantigens.

For a review of intratumoral immunization/in situ vaccination strategiesthat are among those that could be used, see Pierce et al, HumanVaccines & Immunotherapeutics 11(8):1901-1909, 2015; and Marabelle etal, Clin. Cancer Res. 20(7):1747-56, 2014; and Morris et al, Cancer Res;76(13); 3929-41, 2016; all of which are incorporated by referenceherein. In the non-limiting examples disclosed herein, intratumoralimmunization was performed by injecting a fusion protein of an anti-GD2mAb and interleukin 2 (hu14.18-4L2). However, the disclosed methods arenot in any way limited by these examples.

B. Systemically-Administered Immunotherapy: Immune Checkpoint Inhibitorsas Exemplary Immunostimulatory Agents.

In direct contrast to intratumoral immunization by in situ vaccination,systemically-administered immunotherapy is performed by administering animmunostimulatory agent systemically. The immunostimulatory agentcirculates through the whole body of the subject, stimulating the body'snatural immune response.

Immune checkpoint inhibitors are non-limiting examples of suchimmunostimulatory agents. Activated T cells express multiple immuneco-inhibitory receptors, such as lymphocyte-activation gene 3 (LAG-3),programmed cell death protein 1 (PD-1), and cytotoxicT-lymphocyte-associated protein 4 (CTLA4). These and other immunecheckpoint molecules have been shown to modulate T cell responses totumor antigens in the tumor microenvironment through unique andnon-redundant pathways.

More specifically, cancer growth is partly mediated by immunesuppression induced by cancers. Tumors can activate suppressive immunecheckpoint pathways in order to diminish the general immune response tothe tumor. Accordingly, blockade of key immune checkpoint pathways caninduce anti-tumor immunity, facilitated by the patient's own immunesystem.

CTLA4 was the first immune checkpoint molecule to be clinicallytargeted, by administering CTLA4-targeting (anti-CLA4) mAbs. To date,the most promising immune checkpoint inhibitor strategies for thetreatment of cancers involve administering mAbs targeting CTLA-4 and/orPD-1/PD-L1. Other immune checkpoint inhibitor strategies are currentlyin development, and the disclosed combination method is not limited totargeting any specific immune checkpoint pathway.

A series of reviews covering checkpoint inhibitors and cancerimmunotherapy was recently published in volume 276 of ImmunologicalReviews. These reviews, including the introductory overview, Sharpe, A.H., “Introduction to checkpoint inhibitors and cancer immunotherapy,”Immunol Rev. 276 (4 Mar. 2017): 5-8, are incorporated by referenceherein in their entirety.

C. Immunomodulatory Dose of a Radioactive Phospholipid Metal ChelateCompound

The radioactive phospholipid metal chelate compound used shouldselectively target a wide range of solid tumor cell types, such that theRT emitted by the metal isotope chelated to the metal chelate compoundis directed to malignant solid tumor tissue without substantiallyexposing other tissue types to the emitted RT. The radioactive metalisotope included in the radioactive phospholipid metal chelate compoundmay be any radioactive metal isotope known to emit ionizing RT in a formthat would result in immunostimulation of the cells that take up thecompound. Non-limiting examples of radioactive metal isotopes that couldbe used include Lu-177, Y-90, Ho-166, Re-186, Re-188, Cu-67, Au-199,Rh-105, Ra-223, Ac-225, Pb-212, or Th-227.

The immunomodulatory RT dose (as opposed to injected dose) of theradioactive phospholipid metal chelate compound is much less than thedose that would be used for conventional RT against malignant solidtumors. Specifically, the dose should be sufficient to stimulate aresponse in immune cells within the tumor microenvironment (likely byreducing immune-suppressing Treg levels and other immunosuppressivecells or molecules), while not ablating the desired immune cells thatare responsible for the in situ vaccine effect.

The proper immunomodulatory dose can be calculated from imaging dataobtained after administering a “detection-facilitating” dose of aradioactive metal chelate compound. The detection-facilitating dose maybe quite different than the immunomodulatory dose, and the radioactivemetal isotope that is chelated into the radioactive metal chelatecompound may be different (although the rest of the compound structureshould be the same). The radioactive metal isotope used in the detectionstep and dosimetry calculations may be any radioactive metal isotopeknown to emit RT in a form that is readily detectable by conventionalimaging means. Non-limiting examples of “conventional imaging means”include gamma ray detection, PET scanning, and SPECT scanning.Non-limiting examples of radioactive metal isotopes that could be usedinclude Ga-66, Cu-64, Y-86, Co-55, Zr-89, Sr-83, Mn-52, As-72, Sc-44,Ga-67, In-111, or Tc-99m.

D. Metal Chelates of PLE Analogs

The disclosed structures utilize an alkylphosphocholine (APC) carrierbackbone. Once synthesized, the agents should harbor formulationproperties that render them suitable for injection while retaining tumorselectivity as was demonstrated previously with the relatedradiohalogenated compounds. The disclosed structures include a chelatingmoiety to which the radioactive metal isotope will chelate to producethe final imaging or therapeutic agent.

E. Methods of Synthesizing Exemplary M-PLE Analogs

Proposed synthesis of compound 1 is shown below. The first step of thesynthesis is similar to described in Org Synth, 2008, 85, 10-14. Thesynthesis is started from cyclen which is converted into DO3A tris-Bnester. This intermediate is then conjugated with NM404 in the presenceof the base and Pd catalyst. Finally, benzyl protecting groups areremoved by the catalytic hydrogenation.

Synthesis of compound 2 is shown below. It begins with DO3A tris-Bnester which is alkylated with 3-(bromo-prop-1-ynyl)-trimethylsilane.After alkylation, the trimethylsilyl group is removed and theintermediate acetylene is coupled with NM404 by the Sonogashirareaction. The benzyl groups are removed and the triple bond ishydrogenated simultaneously in the last step of the synthesis.

Compounds 5 and 6 can be synthesized from same precursors, DTPAdianhydride and 18-p-(3-hydroxyethyl-phenyl)-octadecyl phosphocholine asshown in the schemes below.

NOTA-NM404 conjugates can be synthesized in an analogous manner. Oneexample of NOTA-NM404 conjugate 7:

E. Dosage Forms and Administration Methods

In situ vaccination can be performed by intratumoral injection, butother administration can apply (topical or systemic). For thesynergistic targeted RT, any route of administration may be suitable. Inone embodiment, the disclosed alkylphosphocholine analogs may beadministered to the subject via intravenous injection. In anotherembodiment, the disclosed alkylphosphocholine analogs may beadministered to the subject via any other suitable systemic deliveries,such as parenteral, intranasal, sublingual, rectal, or transdermaladministrations.

In another embodiment, the disclosed alkylphosphocholine analogs may beadministered to the subject via nasal systems or mouth through, e.g.,inhalation.

In another embodiment, the disclosed alkylphosphocholine analogs may beadministered to the subject via intraperitoneal injection or IPinjection.

In certain embodiments, the disclosed alkylphosphocholine analogs may beprovided as pharmaceutically acceptable salts. Other salts may, however,be useful in the preparation of the alkylphosphocholine analogs or oftheir pharmaceutically acceptable salts. Suitable pharmaceuticallyacceptable salts include, without limitation, acid addition salts whichmay, for example, be formed by mixing a solution of thealkylphosphocholine analog with a solution of a pharmaceuticallyacceptable acid such as hydrochloric acid, sulfuric acid,methanesulfonic acid, fumaric acid, maleic acid, succinic acid, aceticacid, benzoic acid, oxalic acid, citric acid, tartaric acid, carbonicacid or phosphoric acid.

Where the disclosed alkylphosphocholine analogs have at least oneasymmetric center, they may accordingly exist as enantiomers. Where thedisclosed alkylphosphocholine analogs possess two or more asymmetriccenters, they may additionally exist as diastereoisomers. It is to beunderstood that all such isomers and mixtures thereof in any proportionare encompassed within the scope of the present disclosure.

The disclosure also includes methods of using pharmaceuticalcompositions comprising one or more of the disclosed alkylphosphocholineanalogs in association with a pharmaceutically acceptable carrier.Preferably these compositions are in unit dosage forms such as tablets,pills, capsules, powders, granules, sterile parenteral solutions orsuspensions, metered aerosol or liquid sprays, drops, ampoules,auto-injector devices or suppositories; for parenteral, intranasal,sublingual or rectal administration, or for administration by inhalationor insufflation.

For preparing solid compositions such as tablets, the principal activeingredient is mixed with a pharmaceutically acceptable carrier, e.g.conventional tableting ingredients such as corn starch, lactose,sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalciumphosphate or gums, and other pharmaceutical diluents, e.g. water, toform a solid preformulation composition containing a homogeneous mixturefor a compound of the present invention, or a pharmaceuticallyacceptable salt thereof. When referring to these preformulationcompositions as homogeneous, it is meant that the active ingredient isdispersed evenly throughout the composition so that the composition maybe easily subdivided into equally effective unit dosage forms such astablets, pills and capsules. This solid pre-formulation composition isthen subdivided into unit dosage forms of the type described abovecontaining from 0.1 to about 500 mg of the active ingredient of thepresent invention. Typical unit dosage forms contain from 1 to 100 mg,for example, 1, 2, 5, 10, 25, 50 or 100 mg, of the active ingredient.The tablets or pills of the novel composition can be coated or otherwisecompounded to provide a dosage affording the advantage of prolongedaction. For example, the tablet or pill can comprise an inner dosage andan outer dosage component, the latter being in the form of an envelopeover the former. The two components can be separated by an enteric layerwhich, serves to resist disintegration in the stomach and permits theinner component to pass intact into the duodenum or to be delayed inrelease. A variety of materials can be used for such enteric layers orcoatings, such materials including a number of polymeric acids andmixtures of polymeric acids with such materials as shellac, cetylalcohol and cellulose acetate.

The liquid forms in which the alkylphosphocholine analogs may beincorporated for administration orally or by injection include aqueoussolutions, suitably flavored syrups, aqueous or oil suspensions, andflavored emulsions with edible oils such as cottonseed oil, sesame oil,coconut oil or peanut oil, as well as elixirs and similar pharmaceuticalvehicles. Suitable dispersing or suspending agents for aqueoussuspensions include synthetic and natural gums such as tragacanth,acacia, alginate, dextran, sodium caboxymethylcellulose,methylcellulose, polyvinylpyrrolidone or gelatin.

The disclosed alkylphosphocholine analogs are particularly useful whenformulated in the form of a pharmaceutical injectable dosage, includingin combination with an injectable carrier system. As used herein,injectable and infusion dosage forms (i.e., parenteral dosage forms)include, but are not limited to, liposomal injectables or a lipidbilayer vesicle having phospholipids that encapsulate an active drugsubstance. Injection includes a sterile preparation intended forparenteral use.

Five distinct classes of injections exist as defined by the USP:emulsions, lipids, powders, solutions and suspensions. Emulsioninjection includes an emulsion comprising a sterile, pyrogen-freepreparation intended to be administered parenterally. Lipid complex andpowder for solution injection are sterile preparations intended forreconstitution to form a solution for parenteral use. Powder forsuspension injection is a sterile preparation intended forreconstitution to form a suspension for parenteral use. Powderlyophilized for liposomal suspension injection is a sterile freeze driedpreparation intended for reconstitution for parenteral use that isformulated in a manner allowing incorporation of liposomes, such as alipid bilayer vesicle having phospholipids used to encapsulate an activedrug substance within a lipid bilayer or in an aqueous space, wherebythe formulation may be formed upon reconstitution. Powder lyophilizedfor solution injection is a dosage form intended for the solutionprepared by lyophilization (“freeze drying”), whereby the processinvolves removing water from products in a frozen state at extremely lowpressures, and whereby subsequent addition of liquid creates a solutionthat conforms in all respects to the requirements for injections. Powderlyophilized for suspension injection is a liquid preparation intendedfor parenteral use that contains solids suspended in a suitable fluidmedium, and it conforms in all respects to the requirements for SterileSuspensions, whereby the medicinal agents intended for the suspensionare prepared by lyophilization. Solution injection involves a liquidpreparation containing one or more drug substances dissolved in asuitable solvent or mixture of mutually miscible solvents that issuitable for injection.

Solution concentrate injection involves a sterile preparation forparenteral use that, upon addition of suitable solvents, yields asolution conforming in all respects to the requirements for injections.Suspension injection involves a liquid preparation (suitable forinjection) containing solid particles dispersed throughout a liquidphase, whereby the particles are insoluble, and whereby an oil phase isdispersed throughout an aqueous phase or vice-versa. Suspensionliposomal injection is a liquid preparation (suitable for injection)having an oil phase dispersed throughout an aqueous phase in such amanner that liposomes (a lipid bilayer vesicle usually containingphospholipids used to encapsulate an active drug substance either withina lipid bilayer or in an aqueous space) are formed. Suspension sonicatedinjection is a liquid preparation (suitable for injection) containingsolid particles dispersed throughout a liquid phase, whereby theparticles are insoluble. In addition, the product may be sonicated as agas is bubbled through the suspension resulting in the formation ofmicrospheres by the solid particles.

The parenteral carrier system includes one or more pharmaceuticallysuitable excipients, such as solvents and co-solvents, solubilizingagents, wetting agents, suspending agents, thickening agents,emulsifying agents, chelating agents, buffers, pH adjusters,antioxidants, reducing agents, antimicrobial preservatives, bulkingagents, protectants, tonicity adjusters, and special additives.

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.Indeed, various modifications of the invention in addition to thoseshown and described herein will become apparent to those skilled in theart from the foregoing description and the following examples and fallwithin the scope of the appended claims.

III. Examples Introduction to the Examples

These examples demonstrate the potential of bringing together two verydistinct cutting-edge disciplines in cancer treatment research,capitalizing on an unexpected and very potent synergy. These disciplinesare: 1) systemically administered TRT and 2) locally-directed,antibody-mediated, cancer immunotherapy. The data presented hereinsuggest that powerful synergy results from combining these approaches.Together, these two strategies can be used to destroy visiblemacroscopic tumor in a way that enables the destroyed cancer cells tofunction as a potent in situ vaccine that creates tumor-specific T cellimmunity able to eradicate persistent residual metastatic disease, forany type of solid tumor in any location.

Our ongoing preclinical work has shown that combination oftumor-specific mAb with IL2 (to activate innate immune cells) results inaugmented antibody-dependent cell-mediated cytotoxicity (ADCC) [1,2]; aprocess that has already been translated into clinical benefit forchildren with neuroblastoma [3]. Recent preclinical data demonstratemore potent antitumor efficacy when the mAb-IL2 fusion protein isinjected intratumorally (IT) [4,5]. Remarkably, large tumors that do notrespond to these mAb/IL2 injections and continue growing if treated onlywith local xRT, can be completely eradicated if the xRT is combined withthe mAb/IL2 treatment. Most mice are cured and develop T cell memorythat rejects re-challenge with similar tumor cells [6]; demonstratingthat the combined xRT+mAb/IL2 is acting as a potent “in situ”anti-cancer vaccine.

A key limitation is that if there is another macroscopic tumor presentin these animals when they receive xRT+mAb/IL2 treatment to the primary(first) tumor, the second tumor will continue to grow and, to oursurprise, suppress the immune response, preventing any shrinkage of the1^(st) treated tumor. This “concomitant immune tolerance” results, inpart, from suppressive regulatory T cells (Tregs) in the 2^(nd) tumor.Delivering RT alone to both tumors has minimal anti-tumor effect, butdoes deplete these Tregs. Thus, when first tumors are treated withxRT+mAb/IL2, the addition of RT to the second tumor circumvents thisimmune tolerance, enabling eradication of both tumors [7]. Theseobservations indicate a limitation of in situ tumor vaccination in themetastatic setting, but also suggest a robust capacity of RT to overcomethis limitation.

xRT cannot typically be delivered to all metastatic sites withoutprohibitive normal tissue toxicity and immune suppression. Yet notdelivering xRT to all sites of macroscopic disease may leave inhibitoryimmune lineages intact, which are capable of suppressing the immunologicresponse to our local xRT+mAb/IL2 immunotherapy. What is needed,therefore, is a means to deliver RT to all tumor sites in a cancerpatient in a targeted manner.

We have developed TRT vehicles capable of targeting systemicallyadministered RT to both primary and metastatic cancers. One such TRTagent, ¹³¹I-NM404, an intravenously (IV) administered phospholipid ether(PLE) analog, has shown nearly universal tumor targeting properties inover 60 in vivo cancer and cancer stem cell models. This agent iscurrently being evaluated clinically in multiple imaging and therapytrials [8,9]. A systemic injection of ¹³¹I-NM404 localizes in all tumorsregardless of anatomic location and internally provide sufficient RT toablate intratumoral immunosuppressive pathways that can preventdevelopment of an effective, tumor-eradicating, immune response. Theunique attributes of this approach are the near universal tumortargeting capability of NM404, as well as the ability to deliverimmunomodulatory sub-lethal doses of RT to all tumor sites, somethingthat is not typically feasible with xRT. What is new about this is thatour TRT Agents may immuno-modulate all tumors regardless of anatomiclocation, overcoming concomitant tolerance, which will result in along-term in situ tumor vaccination effect following local xRT followedby injection of a tumor specific mAb+IL2. As an increasing number oftumor specific mAbs are becoming approved for clinical use, thiscombination strategy provides an expanded approach for any tumor typethat can be targeted by a tumor-reactive mAb. Furthermore, the approachcan be readily generalized to all in situ tumor vaccination strategies.

Recently, we have discovered that the iodine in ¹³¹I-NM404 can besubstituted with chelators capable of carrying a wide variety ofmetallic imaging (MRI and PET) and TRT radiotherapy moieties. In theseexamples, we describe how to assess the ability of ¹³¹I-NM404 (and thus,the related metal chelate analogs) to initiate the systemicimmunomodulatory response necessary to enable local combined xRT+mAb/IL2treatment to induce a potent radioimmune-facilitated in situ cancervaccine. A similar approach can be used for combined PLEanalog-delivered TRT with other in situ cancer vaccine methods.

In sum, we disclose herein therapeutic and research processes thatcombine two different methods from seemingly disconnected cancer therapydisciplines into a single unified treatment. The data presented in theseexamples indicate that the two methods can be synergistically combinedto effectively eliminate malignant solid tumors and to prevent tumorrecurrence. The three key concepts underlying this approach are that (A)local xRT+IT mAb/IL2 eradicates an existing single tumor and generatesT-cell memory (an in situ vaccine); (B) unless irradiated, distanttumors cause concomitant immune tolerance, preventing in situ vaccineefficacy; and (C) unlike whole body RT, TRT can localize to all tumors,without severe systemic RT-induced immune suppression. These concepts,together with our data, lead to the conclusion that xRT+IT mAb/IL2 to asubject's primary tumor, plus TRT to eliminate tolerance caused bymetastases and enables effective in situ vaccination to eradicate allmalignant solid tumor-based cancers (primary and metastatic sites).

In Example 1, we present background data from our B78 GD2+ model insupport of the method.

In Example 2, we provide guidance for determining the dose of xRT neededfor optimal in situ vaccine effect to a primary tumor, and the lowestdose of xRT to a distant tumor needed to prevent concomitant immunetolerance.

In Example 3, we provide guidance for determining the ¹³1I-NM404 dosingthat approximates the required dosing of xRT to metastases, asdetermined in Example 2, and subsequently evaluating the effects of that¹³1I-NM404 dose on in vivo immune function. Such guidance can besimilarly applied when using the disclosed radioactive phospholipidmetal chelate compounds as the TRT agent.

In Example 4, we provide guidance for using data from Examples 2 and 3to design/test/develop a regimen of ¹³1I-NM404+local xRT+IT-mAb/IL2 inmice bearing two or more tumors in order to destroy the locally treatedtumors and induce T-cell mediated eradication of all distant tumors.Critical issues of TRT and xRT dose and time are optimized for antitumorefficacy. Again, such guidance can be similarly applied when using thedisclosed radioactive phospholipid metal chelate compounds as the TRTagent.

In Example 5, we provide an exemplary synthesis that also finds use tothe synthesis of analogous compounds chelating radioactive metalisotopes.

In Example 6, we demonstrate that an analog having a chelating agent andchelated metal substituted for the iodine moiety of NM404 (Gd-NM600) istaken up by (and can be imaged in) solid tumor tissue, thus providingproof of concept for using the disclosed metal chelates as a TRT agent.

In Examples 7, 8, 9 and 10, we provide information and specific datafrom experimental studies performed in accordance with the guidance ofExamples 1-4.

In Examples 11 and 12, we demonstrate that additional analogs having achelating agents and chelated metals substituted for the iodine moietyof NM404 are taken up by, and can be imaged in, and can be usedtherapeutically for TRT in a range of solid tumor in vivo models, thusproviding additional proof of concept for using the disclosed metalchelates as TRT agents in the disclosed methods.

In Example 13, we discuss how dosimetry in combination with knownradiosensitivities can be used by the skilled artisan to optimizetreatment dosages for any solid tumors.

In Example 14, we discuss differences and advantages in usingalkylphosphocholine metal chelates in the disclosed methods, rather thanthe iodinated compounds exemplified in Examples 1-4 and 7-10.

In Example 15, we demonstrate that TRT in combination withsystemically-administered immunotherapy, rather than in situvaccination, is also effective is treating solid tumors. Theimmunostimulatory agent that is systemically administered may be animmune checkpoint blocker or inhibitor (in this case, anti-CTLA4).

Example 1: Background Supporting Data

The Sondel lab has shown that tumor-specific mAb+IL2 activates innateimmune cells to mediate ADCC in mice [2], with clinical benefit forchildren with neuroblastoma [3]. In mice, IV administration of thehu14.18-IL2 is more potent than IV administration of anti-GD2 mAb+IL2[2, 10]. This can provide dramatic antitumor effects against very smallrecently established GD2+ tumors or very small microscopic metastases,potentially accounting for the clinical use of this approach in patientsin remission but at great risk for relapse [3]. More potent antitumorefficacy against measurable, macroscopic tumors [i.e. ˜50 mm³ GD2+tumors] can be achieved when the IC is injected intratumorally (IT-IC)rather than IV [4,5].

We are now focusing on ways to provide benefit in the setting of muchlarger, macroscopic tumors. Mice bearing a moderately large (200 mm³)B78 melanoma tumor, established five weeks earlier, show no response toIV-IC, and are slowed in their growth by IT-IC, but the tumors continueto grow. These same 200 mm³ tumors also grow after 12 Gy of xRT. Incontrast, when the IT-IC and xRT are combined, 73% of the animals becometumor-free and appear cured of their disease (FIGS. 2A and 2B). Thesemice then show T-cell mediated rejection of rechallenge with the sametumor (FIG. 2C). Thus IT-IC+xRT synergize, inducing the tumor to becomean “in situ tumor vaccine” [6].

In order to simulate clinical metastases, we inoculate mice with B78 inone flank on d-1, and the other flank at week 2. At week 5, the firsttumor is 200 mm³, and the second is 50 mm³. We anticipated thatxRT+IT-IC would destroy the first tumor and that the resultant T cellresponse would then destroy the second. However, adding IT-IC to the xRThad virtually no effect on either the 50 mm³ tumor or the 200 mm³ tumor(FIG. 3 ). This demonstrated a key limitation to the therapy wedelivered; namely, if there is another tumor present when these micereceive xRT+IT-IC to the first tumor, the second tumor will cause asystemic tumor-specific concomitant immune tolerance effect, preventingany shrinkage of either tumor. Importantly, we have found that local xRT(12 Gy) to the first and second tumor simultaneously, abrogates thistolerance effect, allowing IT-IC to the first tumor to induce an immuneresponse that eradicates both tumors in most mice (FIG. 4 ) [7]. Recentdata, using a Treg depleting mAb (not shown) or transgenic mice thatallow selective Treg depletion (FIG. 4 ) [7], demonstrate that thisimmune tolerance is mediated, in part, by regulatory T cells (Tregs); RTto the first and second tumors partially deplete these Tregs,potentially explaining how irradiating both tumors circumvents thetolerance effect [7].

While local xRT to both the first and second tumors circumventstolerance, clinical metastatic disease is often in several locations.All macroscopic metastatic disease must receive RT to block immunetolerance and enable xRT+IT-IC to effectively eradicate all tumor sites.However, delivery of 12 Gy xRT to all sites of disease may be akin to“total body RT” with major dose-dependent (potentially lethal) toxicityand profound systemic immune suppression.

Previously, the Weichert lab has pioneered the development of TRT, inorder to deliver RT to all systemic tumor sites, while minimizing“off-target” RT to normal tissue (especially marrow and immune tissue).

Based on the finding that tumor cells contain an overabundance ofphospholipid ethers (PLE) [11], we synthesized over thirtyradioiodinated PLE analogs in hopes of identifying analogs that wouldselectively target tumors [12]. One of these, NM404, not only displayednear universal tumor uptake in all but three of over 70 in vivo modelsexamined regardless of anatomic location, including brain metastases andcancer stem cells, but also underwent prolonged selective retention onceit entered tumor cells [8]. These diapeutic PLE analogs are unique inthat they avoid premalignant and inflammatory lesions. Surface membranelipid rafts, which are overexpressed on cancer cells relative to normalcells, serve as portals of entry for PLE's, including NM404, into cancerand cancer stem cells [8]. Radioiodinated NM404 (I-124 and I-131), whichhas now been evaluated in five phase 1 and 2 PET imaging trials andthree phase 1 TRT radiotherapy trials, respectively, affords similartumor uptake and retention properties in over a dozen human cancer types[8]. Excellent tumor uptake in the cancer models relevant to theseexamples (the B78 GD2+ murine melanoma) have been confirmed with¹²⁴I-NM404 PET imaging (FIG. 5 ).

Example 2: Determining Dosages of xRT

Our data suggest these four hypotheses: (1) the dose of xRT we have usedto treat a single tumor causes modest direct in vivo tumor death andincreases susceptibility to immune mediated death (via both ADCC and Tcells); (2) the strong T-cell response provided by the addition ofIT-IC, but not IT mAb, suggests that mAb binding to radiated tumorcells, in the presence of IL2, facilitates antigen presentation andaugmented induction of adaptive immunity; (3) the presence of a secondtumor prevents the xRT+IT-IC to the first tumor from causing virtuallyany anti-tumor effect, due to tolerance caused largely by the systemicactions of immunosuppressive cells present in the second tumor [such asTregs and possibly myeloid derived suppressor cells (MDSC)]; thistolerance can be circumvented by depletion of Tregs (FIG. 4 ) orirradiating the second tumor (FIG. 3 ); (4) the dose of RT needed at thesecond tumor to circumvent tolerance might be much lower than the xRTdose needed for the first tumor to become an “in situ vaccine” [14].

Optimizing xRT Dose for the Primary (“In Situ Vaccine”) Tumor Site.

Our in vivo studies of xRT+IT-IC have focused on one dose of 12 Gy tothe first tumor. This is based on our data showing that in vitro RTinduces a dose-dependent functional upregulation of Fas on B78 tumorcells (nearing peak at >12 Gy), coupled to our in vivo datademonstrating our in situ vaccine effect of xRT+IT-IC requires mice withfunctional Fas-L (6). We conducted in vivo pilot studies prior toselecting the 12 Gy dose, which showed higher dose (16 Gy) or increasedfractionation flank RT had toxicity (dermatitis, ulceration, and latelimb edema) and no improvement in tumor response. While we chose a 12 Gysingle fraction of xRT for our in vivo studies, as we move towardsclinical translation, it will be beneficial to better understand themechanism of the local xRT effect and its dose requirements, in order tosafely and effectively induce the in situ vaccine effect.

Our mouse data (FIGS. 2A, 2B and 2C) show that we can induce a potentvaccine effect with 12 Gy xRT+IT-IC, even though 12 Gy of xRT alonecauses no shrinkage of the tumor; it merely slows the progressivegrowth. It is contemplated that we might see just as potent an in situvaccine effect using lower doses of RT. To test this, we will evaluate arange of xRT doses (from 4-16 Gy) as a single fraction in mice bearing a˜200 mm³ B78 tumor, followed by our standard IT-IC regimen (50 mcg/d ondays 6-10). We will determine which xRT doses give optimal tumoreradication and T-cell memory, when combined with IT-IC. If doses lowerthan 12 Gy are less toxic and show comparable efficacy, such lower doseswould be better targets for our xRT dose to the “in situ vaccine” sitein Examples 3 and 4. Similar approaches may be used to optimize dosingfor particular targets or subjects.

Optimizing xRT Dose at a Distant Tumor to Prevent Tolerance fromBlocking “In Situ Vaccination.”

Treating both the first and second tumors with 12 Gy (FIG. 3 ) enablesIT-IC to the first tumor to induce a potent response that eradicatesboth tumors. Our goal is to be able to accomplish this same in situvaccine effect by providing xRT+IT-IC to a single tumor while using theminimal RT dose necessary at metastatic sites to circumvent tolerance.We recognize that xRT itself, especially if widespread, can bemyelo/immunosuppressive. This is why we are pursuing TRT in Examples 3and 4. Even though it is targeted, TRT does have some systemic deliveryof RT. In order to minimize the systemic immune suppression from TRT, wewish to give as low of a dose of TRT as is needed to effectively inhibitthe tumor-induced immune tolerance, while not causing systemicRT-induced global immune suppression. Therefore, it is best to selectthe lowest dose of xRT needed to be delivered to the distant tumor inorder to enable a higher xRT dose to the first tumor to function as anin situ vaccine when combined with IT-IC to the first tumor.

As an exemplary optimization experiment, mice bearing a 200 mm³ firstB78 tumor and a ˜50 mm³ second B78 tumor will receive 12 Gy of xRT tothe first tumor on day-0 (˜5 weeks after implantation of the first B78tumor). This will be followed by our standard regimen of IT-IC on days6-10. Separate groups of mice will receive varying doses of xRT to thesecond tumor. Based on data from the lab of B. Johnson demonstratingthat a total body xRT of 3 Gy can prevent an immunosuppressive effect ina myeloma model (15), we will evaluate doses of 0, 1, 5 and 8 Gy (inaddition to the 12 Gy dose we know is effective). We will see if dosessubstantially less than 12 Gy to the second tumor can be as effective asthe full 12 Gy dose at eliminating the immune tolerance.

Once we have selected the critical dose of xRT where we lose thebeneficial effect, we will perform subsequent analyses to betteroptimize the critical dose. For example, if 5 Gy is as effective as 12Gy, but 1 Gy is not much better than 0 Gy, we would then compare 2, 3,and 4 Gy to identify the critical lowest effective RT dose needed toeliminate tolerance and obtain efficacy in this two tumor model,receiving 12 Gy+IT-IC to the first tumor.

Repeat studies are then be done to confirm if this lowest effective doseto the second tumor still enables an effective in situ vaccine when thedose to the first is the lowest effective dose in the 1-tumor model(tested in Example 2, above) rather than the 12 Gy dose. In summary, thestudies of Example 2 optimize what the lowest xRT doses are for thefirst and second tumors, without losing the efficacy we havedemonstrated with 12 Gy to both.

Initiating Studies of Required xRT Dose to First and Second Tumors inMice Bearing Tumors Other than B78.

To allow our mouse studies to suggest more clinical generalizability, wewill initiate analyses of RT+IT-IC in additional models of GD2+ tumors.We have published on IT-IC with hu14.18-IL2 IC in AJ mice bearing theGD2+NXS2 neuroblastoma [5]. We are also evaluating IT-IC with this sameIC in C57BL/6 mice bearing the GD2+9464D-GD2 neuroblastoma, and thePanc02-GD2 pancreatic cancer that express GD2 via our insertion of thegene for GD2 synthase. As for Example 2, for each model we willdetermine the lowest effective xRT dose needed to the primary and thesecondary tumors to retain the in situ vaccine effect.

Example 3

Determining Dosage of ¹³¹I-NM404 and Evaluating Effects on ImmuneFunction Dosimetry with TRT and immunesuppression from TRT in C57BL/6mice.

¹³¹I-NM404 has shown selective uptake in vitro in >95% of tumor lines(human and mouse), with poor uptake by non-malignant cells, and withsimilar tumor specificity seen in vivo. This includes selective uptakein vivo with the B78 tumor (FIG. 5 ). In our preliminary dosimetrystudy, we gave ¹²⁴I-NM404 to C57BL/6 mice and characterized the timecourse of TRT exposure by serial PET/CT imaging (as in FIG. 5 ). MonteCarlo dosimetry calculations [16-18] based on this study indicated that˜60 Ci of ¹³¹I-NM404 would be needed to deliver ˜3 Gy to an establishedB78 tumor over a four week period of decay. After those four weeks, theremaining TRT dose to the B78 tumor would be less than 0.25 Gy. We willreplicate the data we obtained in our 2-tumor model using xRT (FIG. 3 ),but use the lowest possible dose of targeted ¹³¹I-NM404 TRT to enableeffective elimination of tumor-induced tolerance at all sites of distantdisease. However, unlike xRT, which delivers all dose in minutes and isthen done, TRT deposits dose over time, depending upon both thebiological and physical half-life of the targeted isotope (8 day t½ for¹³¹I). We want an initial TRT effect at the distant tumor sites toeradicate immune tolerance; however we want the immunosuppressive TRTeffect to then be minimal when we give the IT-IC to induce ADCC and thein situ vaccine anti-tumor effects. This is essential to allow fulltumor destruction at all sites.

Using the dosimetry calculations from our preliminary data, we estimatedthat a dose of 3 Ci of ¹³¹I-NM404, should deliver an equivalent of ˜0.2Gy to the tumor site, a dose that we hypothesized should not beimmunosuppressive and should not prevent lymphocyte-mediated tumordestruction. As noted above, this is the dose we estimated would remainyet to be delivered 28 days after an initial ¹³¹I-NM404 dose of 60 Ci.We thus evaluated groups of mice bearing a single 200 mm³ B78 tumor. Onday 0, all mice got 12 Gy xRT to their tumor, and on days 6-10, all got50 mcg/d of IT-IC. One group also got 3 Ci of ¹³¹I-NM404 on d-0 (˜0.2Gy). FIG. 6 shows that the group receiving the ¹³¹I-NM404 had the samedegree of tumor eradication as the group without ¹³¹I-NM404,demonstrating that this low dose of “residual” TRT in the tumor does notblock immune mediated destruction by the RT+IT-IC in situ vaccine. Wethus hypothesize that if we use an initial dose of 60 Ci of ¹³¹I-NM404TRT on day-22, it would effectively block the tolerogenic effect ofdistant tumors, yet enable xRT on day 0 and IT-IC on days 6-10 (28 dafter the TRT) to the first tumor to function as an in situ vaccine,inducing an adaptive response that then eradicates all tumors.

The experiments outlined in this example optimize the dose relationshipstested in FIG. 6 . In our 1-tumor B78 model, we will test a range ofdoses of ¹³¹I-NM404 TRT to select the best TRT dose that results inenough unwanted systemic immune suppression to interfere with thedesired in situ vaccine effect (and thereby slow or prevent eradicationof the first tumor). This is important to Example 4, as it allows us tomake sure the residual radioactivity of the TRT has decayed to less thanthis value at the time we initiate IT-IC to the first tumor in mice withdistant disease. We will also evaluate the kinetics of the TRT responseafter varying TRT doses to select an optimal time period for how long weshould wait after the “tolerance-preventing TRT dose” is given toanimals with multiple tumors to allow the RT+IT-IC treatment of thefirst tumor to still induce the in situ vaccine effect and eradicate theprimary as well as distant tumors.

Related studies will also look at what dose of TRT, given as singleagent treatment, are most beneficial to cause slowing, versus shrinkage,versus eradication of a single B78 tumor. The dose of TRT that is mostbeneficial to eliminate the tumor-induced immune tolerance will besubstantially less than the TRT dose needed to actually induce completetumor destruction (from the TRT alone).

Finally, once the effects of various optimized doses of TRT aredetermined in the 1-tumor model, we will evaluate the subtleimmune-suppressive effects of TRT, by evaluating sera from these subjectfor their immune response to the human IgG component of the IC. We haveshown that immunocompetent mice generate a readily quantified level ofMouse Anti-Human Antibody (MAHA) following treatment with thesehumanized ICs (19). We will use this as a means of determining at whatdose we are seeing the TRT cause a detectible dose-dependent decrease inthe strength of the murine immune response to gauge the overallimmunosuppressive effects from the systemic doses of RT these mice willreceive from this TRT. The low TRT dose that we will need to block thetumor-induced immune tolerance will cause minimal systemic immunesuppression.

Example 4: Developing an Optimal Regimen of ¹³¹I-NM404+LocalxRT+IT-mAb/IL2 in Mice Bearing Two or More Tumors

Testing the Efficacy of TRT+RT+IT-IC in the 2-Tumor B78 Model.

The dose and timing information learned from the studies outlined inExamples 2 and 3 will provide the information we need to optimize TRTdosing and timing required for efficacy in our 2-tumor model. C57BL/6mice will be inoculated with B78 in the left (L) and right (R) flankssimultaneously. Each tumor should be ˜50 mm³ after two weeks and ˜200mm³ after five weeks. If we assume that our dosimetry calculations inExample 3 suggest that we need to deliver 60 Ci of TRT to approximate 3Gy RT to the second tumor (to block the immune tolerance), our externalbeam xRT studies predict that this dose should have minimal slowingeffect on tumor growth. We would plan to treat different groups of micewith 30, 60 or 90 Ci at the 2 w time point (when the tumors are ˜50mm³). Three weeks later the tumors should be ˜200 mm³; at that time wewill give xRT (dose determined as outlined in Example 2) followed sixdays later (˜28 d after the TRT) by five daily injections of IT-IC tothe tumor in the L flank, to induce the in situ vaccine effect. Controlmice would get no TRT, and only the xRT and IT-IC to the L flank,anticipating no in situ vaccine due to tolerance from the distant tumor.A separate group would get local xRT to both tumors and IT-IC to the Lflank, anticipating eradication of both tumors via the in situ vaccineeffect. Another group get TRT+IT-IC, but without local xRT, anticipatingan incomplete vaccine effect.

Follow-up experiments further evaluate varying doses of TRT andvariations in the timing between the TRT and the local xRT+IT-IC to theprimary tumor (L flank). The readouts will be: (A) eradication of theprimary tumor; (B) eradication of the secondary tumor; and (C) systemicimmune suppression, via ELISA analyses of the MAHA response. Our goal isto identify optimal TRT dose and timing with a particular subject anddisease model, to add to the local xRT+IT-IC regimen that can eradicateboth tumors in most subject, while minimizing systemic immunosuppression(as measured by MAHA response).

Optimizing TRT+xRT+IT-IC in Mice Bearing More than Two B78 Tumors.

This section of Example 4 is most analogous to the relevant clinicalsetting; namely, patients with an injectable tumor that could be used asan in situ vaccine site, but with multiple distant metastases that couldeach be causing tumor-induced immune tolerance. These studies willreplicate the conditions found to be most effective in the first part ofExample 4 (above). The important difference is that these subject willeach have four separate tumors, in L and R flanks, and L and Rpara-scapular regions. The TRT is given at the dose and timing foundmost effective in the studies outlined in the first section of Example4, with xRT+IT-IC subsequently given only to the L-flank lesion. Thegoal here is to select TRT dose and timing issues to enable mosteffective in situ vaccine, because the TRT would effectively eliminatethe tumor-induced immune tolerance caused by the three sites not gettingxRT. The measure of efficacy is elimination of all four tumors in mostsubjects. Modifications in TRT dose and timing are tested in order togenerate an optimized regimen that is most effective. Such a regimenfinds use in the clinic for patients with multiple distant metastases,that could not all be irradiated via external beam, but could beirradiated via TRT, when combined with local xRT+IT-IC to the “in situvaccine” site.

Example 5: Synthesis of Metal Chelated NM600

In this Example, we show the synthetic scheme used to synthesize oneexemplary phospholipid chelate, Gd-NM600. Analogs incorporating variousradioactive isotopes could be synthesized in a similar manner, where theradioactive isotope in questions is substituted for Gd.

Scheme for synthesizing Gd-NM600 (the disclosed radioactive metalisotopes could be substituted for Gd):

Example 6: In vivo Imaging Proof of Concept

In this example, we demonstrate the successful in vivo MRI imaging of atumor, using Gd-NM600 as the MRI contrast agent. The data demonstratesthat the backbone phospholipid and chelating agent are taken up andretained by solid tumors, demonstrating that such chelates incorporatingvarious radioactive metals, as disclosed herein, would exhibit similarproperties

For proof-of-concept in vivo imaging of tumor uptake of the Gd-NM404agent, nude athymic mouse with a flank A549 tumor (non small cell lungcancer) xenograft was scanned. The Gd-NM600 agent (2.7 mg) was deliveredvia tail vein injection. Mice were anesthetized and scanning performedprior to contrast administration and at 1, 4, 24, 48, and 72 hoursfollowing contrast delivery. Imaging was performed on a 4.7T Varianpreclinical MRI scanner with a volume quadrature coil. T1-weightedimages were acquired at all imaging time points using a fast spin echoscan with the following pulse sequence parameters: repetition time(TR)=206 ms, echo spacing=9 ms, echo train length=2, effective echo time(TE)=9 ms, 10 averages, with a 40×40 mm² field of view, 192×192 matrix,10 slices of thickness 1 mm each.

As seen in FIG. 7 , MRI imaging of the tumor was significantly enhancedby 24 hours post-injection.

These results demonstrate that the differential uptake and retention ofalkylphosphocholine analogs is maintained for the metal chelated analogsdisclosed herein. Thus, the disclosed metal chelates can readily beapplied to clinical therapeutic and imaging applications.

Example 7: Experiments Determining the Dose of xRT Needed for Optimal InSitu Vaccine Effect to a Primary Tumor, and the Lowest Dose of xRT to aDistant Tumor Needed to Prevent Concomitant Immune Tolerance

As a follow-up to Examples 1-4, dose titration experiments, evaluating avariety of xRT doses, to mice with 1 or 2 tumors have been performed.The first goal has been to test the dose of xRT needed in mice with onetumor to facilitate synergy and an “in situ vaccine” with IT-IC,tumor-reactive mAb linked to IL2. Initial experiments have confirmed ourprior observation that 12 Gy RT alone does not eradicate or even regressthe growth of established B78 melanoma tumors (0% complete regression),whereas 12 Gy+IT-IC results in complete regression of most B78 tumors(66%) in mice bearing a single tumor. On the other hand, 2 Gy+IT-ICslows tumor progression compared to IT-IC alone (mean tumor size day32=472 mm³ vs 1214 mm³, respectively) but did not render any micedisease free (0% complete regression).

In our “2-tumor model”, we have previously shown that treatment of one“primary” tumor with xRT+IT-IC is not effective in treating either thetreated primary tumor or the untreated “secondary” tumor. In fact, inthis 2-tumor model we have observed that the presence of the secondtumor eliminates the efficacy of IT-IC injection following xRT. We havedesignated this phenomenon as “concomitant immune tolerance” (CIT), anddemonstrated that this results, at least in part, from T regulatorycells (Tregs) in the distant (non-irradiated) secondary tumor, whichcirculate systemically and repopulate the xRT-treated/IT-IC injectedprimary tumor. These Tregs that return to the primary tumor appear tointerfere with the desired “in situ vaccine” effect.

We have now confirmed our prior observation that CIT can be overcome bydelivering 12 Gy xRT to both the primary and the secondary tumor.Importantly, given that Tregs are quite sensitive to RT, we hypothesizedthat a lower dose of RT could be delivered to the secondary tumor inorder to overcome CIT and rescue response to in situ vaccination at theprimary tumor (primary tumor treated with 12 Gy+IT-IC). We have nowtested this and observed that xRT doses of 2 Gy or 5 Gy to the secondarytumor are comparable to 12 Gy in their capacity to blunt CIT and rescueresponse to primary tumor treatment with 12 Gy+IT-IC. These importantexperiments have been repeated in duplicate, and suggest (ashypothesized) that the dose of xRT that must be given to distant tumorsto prevent CIT is much less than the dose needed at the IT-IC injectedprimary tumor site for the purpose of generating an in situ vaccineeffect.

This supports our overarching hypothesis in this disclosure, andsuggests that in animals bearing multiple tumors we will be able todeliver a relatively low dose of RT to all sites of disease using thetargeted radiotherapeutic (TRT) NM600, and thereby overcome CIT whenthis is combined with local xRT and IT-IC injection of a single tumorsite (the in situ vaccine site).

Example 8: Experiments Determining the ¹³¹I-NM404 Dosing thatApproximates the Required Dosing of xRT to Metastases, as DeterminedAbove, and then Evaluating the Effects of that ¹³¹I-NM404 Dose on InVivo Immune Function

Based on the preliminary data described above in Examples 1-4, studieshave been done to move these concepts into in vivo testing using TRT.Dosimetry studies have been performed on mice bearing 1 or 2 B78 tumors(the tumor model that we have used to demonstrate best our in situvaccine approach and the hurdle of CIT). This was done in order toestimate the amount of ¹³¹I-NM404 that would be needed to approximate ˜2Gy of xRT.

In order to then determine if a ˜2 Gy equivalent dose of ¹³¹I-NM404would have the desired effects against intratumor lymphoid cells(particularly Tregs), 2 separate approaches have been pursued. First, weadministered this dose of ¹³¹I-NM404 to mice bearing a radiosensitivelymphoma tumor, which exhibits comparable NM404 uptake to B78 tumors.Following this we have documented potent lymphoid tumorshrinkage/dose-dependent inhibition under conditions that did not causeeither substantial shrinkage/slowing of the B78 tumor or any evidentdepletion of circulating lymphoid cells (as gauged by peripheralcomplete blood counts). These data are consistent with the fact thatlymphoid cells are much more sensitive to low-dose RT than are typicalsolid tumor cells, and suggest that selective uptake of TRT in tumor mayenable intratumor lymphoid cell depletion without systemic lymphopenia.These studies also suggest that such a lymphoid tumor could serve as anin vivo biological “dosimeter” for identifying and monitoring the effectof TRT on intratumor lymphoid cells.

A second approach involved treating mice with B78 tumors with these samedoses of ¹³¹I-NM404. These animals were then sacrificed at half-life (8d) intervals, and after sufficient delay for radioactive decay, thetumors were stained for the presence of effector T cells and Tregs byimmunohistochemistry Intriguingly, the animals receiving ¹³¹I-NM404 inthis initial experiment showed no systemic lymphopenia at any time point(by peripheral complete blood count) but did show a decrease inintratumor FoxP3+ Tregs at 2 half-lifes following TRT administration. Atthis 2-half-life time point, we also observed a decrease in intratumoreffector CD8+ T cells. Importantly, however at subsequent 3 and 4half-life time points we observed an increase in intratumor CD8+effector T cells but a further decline in the levels of intratumorTregs, both compared to untreated baseline and 2^(nd) half-life levels.This observation again supports our hypothesis that it will be feasibleto use TRT to overcome Treg-mediated CIT in order to rescue an in situvaccine effect in animals bearing multiple tumors.

Finally, to characterizing the immunological effects of TRT on theimmune cells within tumors, we have treated B78 bearing mice with¹³¹I-NM404 and collected tumor tissue at pretreatment and at half-life(8 d) intervals thereafter. These tissues were then analyzed by RT-PCRfor gene expression of a panel of immune signatures. The resultsindicate that TRT treatment alone causes striking changes in expressionof tumor cell markers of immune susceptibility and in genes normallyexpressed only by immune cells, with the latter showing a clear timecourse of decreased expression followed by rebound over-expression.

Example 9: Experiments Using Data from Examples 5 and 6 to Develop aRegimen of ¹³¹I-NM404+ Local xRT+IT-mAb/IL2 in Mice Bearing Two or MoreTumors and Induce T-Cell Mediated Eradication of all Distant Tumors

This Example illustrates treating animals bearing tumors in at least 2locations. Our strategy involves using xRT and local IT-IC at the insitu vaccine site, in combination with TRT systemically to inhibit CIT,in order to obtain enhanced anti-tumor immune activity at all tumorsites. Critical issues of TRT and xRT dose and timing will be optimizedfor antitumor efficacy.

Using the data summarized in Examples 7 and 8, a study was done in micebearing 2 separate B78 tumors. Mice received the estimated requiredsystemic ¹³¹I-NM404 dose followed by xRT and local immunotherapy to thein situ vaccine site. With appropriate controls, this dose of ¹³¹I-NM404did appear to attenuate CIT, as desired in mice with 2 tumors. Inaddition, in mice with one tumor, this TRT dose did not appear tointerfere with the local in situ vaccine effect (as hypothesized anddesired). Further testing, and modification of some of the experimentalvariables, is underway in order to try to maximize the desired effect ofblocking CIT without suppressing the in situ vaccine effect. Moredetails regarding these experiments are disclosed in Example 10 below.

Example 10: Data from Mice Bearing Two or More Tumors

Tumor-Specific Inhibition of Primary Tumor Response to the Combinationof Local xRT+IT-IC by a Distant Untreated Tumor in Murine Melanoma andPancreatic Tumor Models.

C57BL/6 mice bearing a syngeneic, GD2+ primary flank tumor +/−asecondary tumor on the contralateral flank were treated to the primarytumor only, as indicated, with xRT on day “1” and IT injection of 50 mcgof the anti-GD2 IC, hu14.18-IL2 on day 6-10.

In mice bearing a primary B78 melanoma tumor, the presence of anuntreated secondary B78 tumor antagonized primary tumor response toxRT+IT-IC (FIG. 8A). We describe this effect as “concomitant immunetolerance”—an antagonistic effect of a non-treated distant tumor on thelocal response of a treated tumor to xRT+IT-IC. Kaplan-Meier survivalcurves were obtained for these mice plus replicate experiments (FIG.8B). Nearly all mice were euthanized due to primary tumor progression.

In mice bearing a primary Panc02-GD2+ pancreatic tumor, with or withouta secondary Panc02-GD2-tumor on the opposite flank, the presence of anuntreated Panc02 secondary tumor suppressed the response of a primaryPanc02-GD2+ tumor to xRT+IT-IC (FIG. 8C). In mice bearing a primary B78melanoma tumor, a secondary B78 tumor suppressed primary tumor responseto xRT+IT-IC but a secondary Panc02-GD2+ pancreatic tumor did not exertthis effect (FIG. 8D). In mice bearing a primary Panc02-GD2+ tumor asecondary Panc02-GD2-tumor suppressed primary tumor response to combinedxRT and IT-hu14.18-IL2, while a B78 secondary tumor did not (FIG. 8E).

Concomitant Immune Tolerance is Circumvented by Specific Depletion ofRegulator T Cells (Tregs).

Immunohistochemistry images were obtained for the Treg marker, FoxP3 fortumors evaluated on day 6 after xRT in mice with one or two tumors (FIG.9A). Mice received no xRT, or xRT only to the primary tumor. DEREG miceexpress diphtheria toxin receptor under control of the Treg-specificFoxP3 promoter, enabling specific depletion of Tregs upon IP injectionof diphtheria toxin (FIGS. 9B and 9C). DEREG mice bearing primary andsecondary B78 melanoma tumors were treated with xRT+IT-IC to the primarytumor and IP injection of either diphtheria toxin or PBS. Concomitantimmune tolerance is eliminated following depletion of Tregs in thesemice, resulting in improved primary (FIG. 9B) and secondary (FIG. 9C)tumor response.

Concomitant Immune Tolerance is Overcome by Delivering xRT to Both TumorSites.

In mice bearing primary and secondary B78 tumors, the secondary tumorsuppresses primary tumor response to primary tumor treatment withxRT+IT-IC. This is overcome by delivering 12 Gy xRT to both the primaryand secondary tumors and IT-IC to the primary tumor, resulting inimproved primary tumor response (FIG. 10A) and aggregate animal survival(FIG. 10B) from replicate experiments.

Low Dose xRT Alone does not Elicit In Situ Vaccination but does OvercomeConcomitant Immune Tolerance when Delivered to Distant Tumor SitesTogether with 12 Gy+IT-IC Treatment of an In Situ Vaccine Site.

In mice bearing a primary B78 tumor only, 12 Gy+IT-IC elicits in situvaccination (as shown previously) and results in complete tumorregression in most mice (FIG. 11A) and a memory immune response (Morris,Cancer Res, 2016). On the other hand no animals exhibit complete tumorregression following either IT-IC alone or low dose (2 Gy) xRT+IT-IC (0/6 in both groups) p<0.05.

In mice bearing a primary and secondary B78 melanoma tumor, low dose xRT(2 Gy or 5 Gy) delivered to the secondary tumor is comparable to 12 Gyin its capacity to overcome concomitant immune tolerance at the primarytumor (FIG. 11B). In these same animals, it is apparent that overcomingconcomitant immune tolerance by delivery of low dose xRT to thesecondary tumor rescues a systemic response to IT-IC immunotherapy (FIG.11C). In this context, when RT is delivered to all tumor sites thenIT-IC injection of the primary tumor triggers a systemic anti-tumoreffect that renders secondary tumor response to 2 Gy or 5 Gy greaterthan the response to 12 Gy RT in absence of primary tumor IT-ICinjection.

Low Dose TRT with ¹³¹I-NM404 Effectively Depletes Tumor InfiltratingFoxP3+ Tregs without Systemic Leukopenia or Depletion of TumorInfiltrating CD8+ Effector T Cells.

In most clinical scenarios, it is not feasible to deliver external beam,even low dose, to all tumor sites without eliciting marked bone marrowdepletion and leukopenia that would result in immunosuppression. Here wetested whether TRT could be administered systemically to specificallydeplete tumor infiltrating suppressive immune cells (Tregs), withouttriggering systemic immune cell depletion and leukopenia. Dosimetrystudies in this B78 melanoma tumor model were performed usingpositron-emitting ¹²⁴I-NM404 confirm tumor-selective uptake of NM404(FIG. 12A). C57BL/6 mice bearing B78 tumors were treated with 60 μCi131I-NM404. This activity approximates the amount of ¹³¹I-NM404necessary to deliver ˜2 Gy TRT to a B78 tumor. Peripheral blood andtumor samples were collected in untreated control mice (C) and at 8 dayintervals (T1=d8, T2=d16, T3=d24, T4=d32) thereafter. This dose of TRTdid not result in any significant systemic leukopenia (FIG. 12B) and didnot significantly affect the level of tumor infiltrating CD8+ effector Tcells (FIG. 12C). However, tumor infiltrating FoxP3+ Tregs weresignificantly depleted by this dose of TRT (FIG. 12D).

Low Dose TRT with ¹³¹I-NM404 Effectively Overcomes Concomitant ImmuneTolerance and Rescues the Systemic Anti-Tumor Effect of In SituVaccination.

Given the capacity of low dose 131I-NM404 TRT to depletetumor-infiltrating Tregs without rendering a mouse leukopenic, we testedwhether low dose 131I-NM404 might effectively overcome concomitantimmune tolerance. C57BL/6 mice bearing two B78 tumors were treated with60-μCi ¹³¹I-NM404 on day 1 (NM404), as indicated. After one half-life(day 8), animals received 12 Gy xRT or no xRT to the primary tumor (insitu vaccine site). Control mice receiving no ¹³¹I-NM404 were treated tothe secondary tumor as indicated (0, 2, or 12 Gy). Mice received dailyIT injections of IC to the primary tumor (in situ vaccine site), asindicated, on days 13-17. Primary tumor (FIG. 13A) and secondary tumor(FIG. 13B) response demonstrates that administration of low dose TRTeffectively overcomes concomitant immune tolerance and rescues thesystemic anti-tumor effect of in situ vaccination.

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Science Translational Medicine 6, 240ra75,    1-10. 2014.-   Morris Z S, JP Weichert, J Sakera, EA Armstrong, A Besemer, B    Bednarz, R Kimple, P M Harari. Therapeutic combination of    radiolabeled NM404 with external beam radiation in head and neck    cancer model systems. Radiotherapy and Oncology. J. Radiation    Oncology, DOI: 10.1016. 2015.-   Lode H N, Xiang R, Dreier T, Varki N M, Gillies S D, Reisfeld R A.    Natural killer cell-mediated eradication of neuroblastoma metastases    to bone marrow by targeted interleukin-2 therapy. Blood 91(5),    1706-1715. 1998.-   Snyder F, Wood R. Alkyl and alk-1-enyl ethers of glycerol in lipids    from normal and neoplastic human tissues. Cancer Res 29, 251-257.    1969.-   Pinchuk A N, Rampy M A, Longino M A, Skinner R W, Gross M D,    Weichert J P, Counsell R E, Synthesis and structure-activity    relationship effects on the tumor avidity of radioiodinated    phospholipid ether analogues. J Med Chem 49, 2155-2165. 2006.-   Swanson K I, Clark P A, Pinchuk A N, Longino M A, Farhoud M,    Weichert J P, Kuo J S. Initial Studies on Novel Cancer-Selective    Alkylphosphocholine Analogs CLR1501 and CLR1502 for    Fluorescence-guided Neurosurgery. Neurosurgery. 76(2): 115-123.    2015.-   Filatenkov A, Baker J, Mueller A M, Kenkel J, Ahn G O, Dutt S, Zhang    N, Kohrt H, Jensen K, Dejbakhsh-Jones S, Shizuru J A, Negrin R N,    Engleman E G, Strober S. Ablative Tumor Radiation Can Change the    Tumor Immune Cell Microenvironment to Induce Durable Complete    Remissions. Clin Cancer Res. 21:3727-39. 2015.-   Jing W, Gershan J A, Weber J, Tlomak D, McOlash L, Sabatos-Peyton C,    Johnson B D. Combined immune checkpoint protein blockade and low    dose whole body irradiation as immunotherapy for myeloma. J    Immunother Cancer. 3:2. 2015.-   Bednarz B., Besemer A., Yang Y. A Monte Carlo-Based Small Animal    Dosimetry Platform for Pre-Clinical Trials: Proof of Concept. Med.    Phys. 39, 3899. 2012.-   Besemer et al. Towards Personalized Dosimetry Using Diapeutic    Radiopharmaceuticals. Med. Phys. 40, 382. 2013.-   Besemer A. and Bednarz B. Validation of a patient-specific Monte    Carlo targeted radionuclide therapy dosimetry platform. Med. Phys.    41, 303. 2014.-   Imboden M, Murphy K R, Rakhmilevich A L, Neal Z C, Xiang R, Reisfeld    R A, Gillies S D and Sondel P M. The level of MHC Class I expression    on murine adenocarcinoma can change the antitumor effector mechanism    of immunocytokine therapy. Cancer Res. 61:1500-7. 2001.

Example 11

In Vivo Uptake of Multiple N M600 Metal Chelates in Mice Xenograftedwith Eight Different Solid Tumor Types, Demonstrated by PET Imaging

In this example, we demonstrate the differential uptake of NM600chelated with four different metals in a range of solid tumors in vivo,as demonstrated by PET/CT imaging of such tumors. These data provideadditional support for the use of metal chelated alkylphosphocholineanalogs as TRT agents for eliminating tumor-induced immune tolerance, asdisclosed herein. The structure of NM600 is shown in FIG. 14 , as anexample species chelated with ⁶⁴Cu (⁶⁴Cu-NM600); however, any metal canbe readily chelated to NM600.

Specifically, mice were each xenografted with one of eight differentsolid tumor cell lines (B78 (melanoma), U87MG (glioblastoma), 4T1(breast carcinoma), HCT-116 (colorectal carcinoma), A549 (lungcarcinoma), PC-3 (prostate carcinoma), HT-29 (colorectaladenocarcinoma), or MiaPaca (pancreatic carcinoma). For each of thexenografted mice, cell suspension containing tumor cells was inoculatedinto subcutaneous tissue of one or both flanks of the mouse. Oncexenograft tumors reached a minimum size, each mouse was injected withbetween 150-300 μCi of NM600 radiolabeled with ⁶⁴Cu, ⁸⁹Zr, ⁸⁶Y, or ⁵²Mnvia lateral tail vein injection. After an uptake period, PET imaging wasperformed in an Inveon micro PET/CT. Right before each scan, mice wereanesthetized with isoflurane (2%) and placed in a prone position in thescanner. Longitudinal 40-80 million coincidence event static PET scanswere acquired at 3, 12, 24, and 48 hours post-injection of theradiotracer and the images were reconstructed using an OSEM3D/MAPreconstruction algorithm.

FIG. 15 shows the resulting images 48 hours post-injection-forsingle-tumor B78 mice injected with ⁸⁶Y-NM600; FIG. 16 shows theresulting images 48 hours post-injection-for two-tumor B78 mice injectedwith ⁸⁶Y-NM600; FIG. 17 shows the resulting images 3, 24 and 48 hourspost-injection for a U87MG mouse injected with ⁶⁴Cu-NM600; FIG. 18 showsthe resulting images 3, 24 and 48 hours post-injection for a 4T1 mouseinjected with ⁶⁴Cu-NM600; FIG. 19 shows the resulting images 3, 24 and48 hours post-injection for an HCT-116 mouse injected with ⁶⁴Cu-NM600;FIG. 20 shows the resulting images 3, 24 and 48 hours post-injection foran A549 mouse injected with ⁶⁴Cu-NM600; FIG. 21 shows the resultingimages 3, 24 and 48 hours post-injection for a PC-3 mouse injected with⁶⁴Cu-NM600; FIG. 22 shows the resulting images 3, 24 and 48 hourspost-injection for a HT-29 mouse injected with ⁶⁴Cu-NM600; FIG. 23 showsthe resulting images 3, 24 and 48 hours post-injection for a MiaPacamouse injected with ⁶⁴Cu-NM600; FIG. 24 shows the resulting images 3, 24and 48 hours post-injection for a 4T1 mouse injected with ⁸⁶Y-NM600;FIG. 25 shows the resulting images 3, 24 and 48 hours post-injection fora 4T1 mouse injected with ⁸⁹Zr-NM600.

For HT-29 and PC3 mice injected with ⁵²Mn-NM600, PET images wereobtained at 4 hours, and one day post-injection (FIG. 26 for HT-29; FIG.27 for PC3), as well as on days 2, 3, 5 and 7 post-injection (FIG. 28for HT-29; FIG. 29 for PC-3).

As seen in FIGS. 15-29 , the scanned mice produced PET/CTthree-dimensional volume renderings showing cumulative absorbed dosedistribution concentrated in the xenografted tumor. The results confirmthe differential uptake of metal chelated NM600 into the xenograftedsolid tumor tissue, and demonstrate the potential use of NM600 analogsincorporating radioactive metal isotopes in the disclosed treatmentmethods.

Quantitative region-of-interest analysis of the images was performed bymanually contouring the tumor and other organs of interest. Quantitativedata was expressed as percent injected doe per gram of tissue (% ID/g).Exemplary data show that 4T1 tumor tissue increased its uptake over timeand effectively retained all three tested NM600 chelates (⁸⁶Y-NM600,⁶⁴Cu-NM600 and ⁸⁹Zr-NM600, see FIG. 30 ), while healthy heart (FIG. 31), liver (FIG. 32 ) and whole body tissue (FIG. 33 ) all exhibitedsignificantly decreased uptake/retention over time.

Ex vivo biodistribution analysis was performed after the lastlongitudinal PET scan. Mice were euthanized and tissues harvested,wet-weighed, and counted in an automatic gamma counter (Wizard 2480,Perkin Elmer). Exemplary biodistribution data show significant uptakeand retention in tumor tissue (4T1) for different NM-600 chelates(⁸⁶Y-NM600, ⁶⁴Cu-NM600, ⁸⁹Zr-NM600 and ¹⁷⁷Lu-NM600, see FIG. 34 ),

Together, these results demonstrate that the disclosed metal chelatescan readily be used for the TRT step of the disclosed treatment methods.

Example 12

Demonstrating Anti-Tumor Activity and Tumor Autoradiography with TwoDifferent N M600 Metal Chelates Against Multiple Solid Tumor Types inXenografted Mice

In this example, using three different solid tumor models, we show thatalkylphosphocholine metal chelate analogs can be effectively used tofacilitate conventional TRT. These results further demonstrate thepotential for using the metal chelates in the TRT step of the presentlydisclosed treatment methods.

B78, MiaPaca and 4T1 subcutaneous flank xenografts were induced in mice,as described previously. Subsequently, the mice were administeredtherapeutic doses (250-500 μCi) of ⁹⁰Y-NM600, ¹⁷⁷Lu-NM600, or a controlsolution via lateral tail vein injection.

Planar 2D phosphor images of the biodistribution of the agent were takenusing a Cyclone Phosphorimager (Perkin Elmer). Mice were anesthetizedand place in direct contact with the phosphor plate in a supineposition, where they remained for a period of 15-30 min; plates werethen read in the phosphorimager. Various images were recorded between 4and 96 h post-injection of the radioactive dose. The resultingautoradiography images demonstrate rapid and selective uptake and longterm retention of the chelates in all of the solid tumor tissues typestested (see FIGS. 40, 41, 42, 43, 44 and 45 ).

Tumor response was assessed by comparing tumor growth of the treated vs.control mice. Tumor volume was determined by measuring tumor's lengthand width with calipers and calculating the volume using the formula forthe volume of the ellipsoid. Mice weight was also recorded. Humaneendpoints were defined as: tumor volume>2500 m³ or significant weightlost below 13 g.

As seen in FIGS. 46, 47, 48, 49, 50 and 51 , the results demonstratethat the two tested NM600 chelates had a statistically significant invivo therapeutic effect when compared with the control, resulting indecreased mean tumor volumes for double doses of ¹⁷⁷Lu-NM600 in 4T1xenografts (see FIG. 50 ), and reducing growth to near zero or slowingthe growth rate of MiaPaca, 4T1 or B78 xenografts given a single dose of¹⁷⁷Lu-NM600 (see FIGS. 47, 48, and 49 ) or B78 or 4T1 xenografts given asingle dose of ⁹⁰Y-NM600 (see FIGS. 46 and 51 ).

These results further demonstrate the efficacy of using the disclosedalkylphosphocholine metal chelates to deliver TRT to effectively treatsolid tumors of various types.

Example 13 Coupling Radiation Dosimetry and Radiosensitivity Index toPredict TRT Response in a Wide Range of Solid Tumor Types

In this example, we discuss factors for determining chelate dosagesappropriate for the TRT step of the disclosed methods in a range ofsolid tumor types.

Estimation of Tumor Absorbed Doses

Whether the amount of ¹⁷⁷Lu/⁹⁰Y-NM600 that is administered isimmunostimulatory or cytotoxic depends on the tumor absorbed dose. Thediapeutic property of NM600, that ⁶⁴Cu/⁸⁶Y-NM600 can be used as animaging surrogate for therapeutic metals ¹⁷⁷Lu/⁹⁰Y-NM600, respectively,was leveraged to estimate tumor dosimetry. Ultimately, ⁶⁴Cu/⁸⁶Y-NM600PET/CT was used to quantitatively measure in vivo biodistribution andestimate radiation dosimetry which can help identify dose limitingorgans and potential tumor efficacy of ¹⁷⁷Lu/⁹⁰Y-NM600 TRT.

The general concept is as follows: (1) the concentration of⁶⁴Cu/⁸⁶Y-NM600 within the tumor is quantified over time usinglongitudinal PET/CT imaging, (2) the concentration of ⁶⁴Cu/⁸⁶Y-NM600 isdecay corrected to account for the difference in decay rates between the⁶⁴Cu/⁸⁶Y-NM600 and ¹⁷⁷Lu/⁹⁰Y-NM600, (3) the concentration of¹⁷⁷Lu/⁹⁰Y-NM600 within the tumor is time-integrated to compute thecumulative activity, or total number of decays, (4) the deposition ofthe radionuclide decays is modeled within the tumor and quantified.

Steps (1) through (3) can be performed with any medical image processingsoftware package whereas step (4) requires sophisticated radiationdosimetry software. OLINDA/EXM (Stabin, Sparks and Crowe 2005) is adosimetry estimation software with 510(k) approval that uses theformalism developed by the Medical Internal Radiation Dose (MIRD)committee of the Society for Nuclear Medicine (Bolch et al., 2009). TheMIRD approach estimates the mean absorbed dose received by a tissue ororgan due to the radiation emitted from within the organ itself or fromanother source organ. The simplest form of the MIRD equation,

D(t←s)=Ã _(S) S(t←s),

gives the absorbed dose, D[mGy], to a target region t from theradionuclide activity within a source region s. The radionuclideactivity of s is expressed as a cumulated activity Ã_(s) which is thetotal number of radionuclide decays given in units of MBq-s. TheS-factor, S(t←s) [mGy/MBq-s], is the fraction of the energy released byone radionuclide decay within the source region s which is depositedwithin the target region t normalized by the mass of the target regiont, m_(t). The S-factor is a tabulated value calculated using Monte Carloin a set of standard phantoms and organs. Typically, we are concernedwith the dose per unit of injected activity, D[mGy/MBq]. The equation iswritten in terms of the residence time, τ_(h),[MBq-s/MBq_(inj)],

${\tau_{h} = \frac{{\overset{\sim}{A}}_{s}}{A_{inj}}},$

which is the ratio of the cumulative activity and the injectionactivity, A_(inj)[MBq], as

${\overset{¯}{D}\left( t\leftarrow s \right)} = {\frac{D\left( t\leftarrow s \right)}{A_{inj}} = {{\left( \frac{{\overset{\sim}{A}}_{s}}{A_{inj}} \right) \cdot S} = {\tau_{h} \cdot {S.}}}}$

In the case of calculating tumor dosimetry, OLINDA/EXM models the tumoras an isolated unit density sphere whose volume was estimated from thetumor region of interest (ROI) created as part of step (1). Theconcentration of NM600 (% ID/g) within the tumor was determined at eachtime point and decay corrected. Cumulative activity was then calculatedby integrating the concentrations over all time using trapezoidalpiecewise integration.

Radiation dosimetry results for many cell lines are shown in Table 1.This information can be used to estimate the absorbed dose forradiotherapy studies aimed to either eradicate tumors or stimulate theimmune system.

TABLE 1 Dosimetry estimates for both ¹⁷⁷Lu-NM600 and ⁹⁰Y-NM600(Gy/MBq_(inj)) using either ⁶⁴Cu-NM600 or ⁸⁶Y-NM600 PET imaging as asurrogate PC3 A549 HT-29 MiaPaca U87MG 4T1 B78 Lu-177 0.39 0.30 0.490.24 0.58 1.50 0.92 Y-90 0.69 0.53 0.84 0.45 1.01 4.68 2.86

Radiosensitivity Index to Predict Dose-Response

Intrinsic radiosensitivity is a crucial factor underlying radiotherapyresponse; and, knowing it apriori for a cancer type could help predicthow it may respond to radiation from TRT. However, since there is nomethod for its routine assessment in tumors, radiosensitivity ismeasured as the surviving fraction (between 0 and 1) followingirradiation with 2 Gy (SF2) by clonogenic assay. The relativeradiosensitivity of cancer cell phenotypes ranges from those that havevery low radiosensitivities (pancreas, colorectal, glioma and breast) tothose with high radiosensitivities (lymphomas). Cancers can becategorized or ranked by their radiosensitivity indices (Table 2).

If we can demonstrate good tumor uptake and growth inhibition with APCmetal chelates in a highly radiosensitive tumor like lymphoma and in ahighly radiation resistant tumor like glioma, breast, pancreatic orcolorectal, then it can be implied that these agents would be effectiveagainst any tumor with an SF2 value between that of lymphoma and glioma(0.3-0.82) if they are able to target the tumor in vivo. It would alsobe expected then that the radiation dose needed to eradicate gliomatumor cells would be higher than that needed to treat the moreradiosensitive lymphoma cells.

We currently have in vivo imaging to confirm tumor selectivity andtherapy response (tumor growth inhibition) data in all the tumor celllines listed in Table 2. In some cases, it may be necessary to givemultiple doses of the APC chelates to elicit sufficient cancer cellkill. By using quantitative imaging coupled with radiation dosimetrycalculation, we can estimate the tumor absorbed dose necessary to eitherkill the cancer cells (higher doses) or stimulate the immune system, asdisclosed herein (lower doses).

Coupling dosimetry estimates for a variety of cancer cell lines(Table 1) with their respective radiosensitivity indices (Table 2)supports the establishment of a dose response landscape for NM600. Byknowing the tumor targeting characteristics and efficacy of NM600 withina series of cell lines, it is possible to estimate the absorbed tumordose and potential efficacy of cell lines with similar radiosensitivityindices. Furthermore, treatment doses can be linearly scaled accordingto Table 1, depending on the desired outcome of tumor eradication orimmuno-stimulation (as disclosed herein).

TABLE 2 Relative Radiosensitivity of Cancer Cells Imaging uptake and orgrowth inhibition with Tumor Type Cell Line SF2 value APC chelates Refs.Breast MDA-MB-231 0.82 Yes 8 Pancreatic Mia-Paca 0.80 Yes 6, 7Colorectal HCT-29 0.75 Yes 7 Melanoma B-78 0.65 Yes 3, 4, 7 Glioma(brain) U-87 0.63 Yes 1, 2, 7 Lung A-549 0.61 Yes 5, 7 (NSCLC) ProstatePC-3 0.55 Yes 4 Lymphoma EL-4 0.30 Yes 3, 7 SF₂ = surviving fractionfollowing exposure to 2 Gy of in vitro radiation exposure * Several celllines ¹Taghian, Alphonse, et al. “In vivo radiation sensitivity ofglioblastoma multiforme.” International Journal of Radiation Oncology*Biology* Physics 32.1 (1995): 99-104. ²Ramsay, J., R. Ward, and N. M.Blechen. “Radiosensitivity testing of human malignant gliomas.”International Journal of Radiation Oncology* Biology* Physics 24.4(1992): 675-680. ³Fertil, B., and E. P. Malaise. “Intrinsicradiosensitivity of human cell lines is correlated withradioresponsiveness of human tumors: analysis of 101 published survivalcurves.” International Journal of Radiation Oncology* Biology* Physics11.9 (1985): 1699-1707. ⁴Wollin, Michael, et al. “Radio sensitivity ofhuman prostate cancer and malignant melanoma cell lines.” Radiotherapyand Oncology 15.3 (1989): 285-293. ⁵Kodym, Elisabeth, et al. “Thesmall-molecule CDK inhibitor, SNS-032, enhances cellularradiosensitivity in quiescent and hypoxic non-small cell lung cancercells.” Lung Cancer 66.1 (2009): 37-47. ⁶Unkel, Steffen, Claus Belka,and Kirsten Lauber. “On the analysis of clonogenic survival data:Statistical alternatives to the linear-quadratic model.” RadiationOncology 11.1 (2016): 11. ⁷E P Malaise, Patrick J. Deschavanne, andBernard Fertil, “Intrinsic radiosensitivity of human cells.” Advances inradiation biology 15 (2016): 37-70. ⁸Siles, E., et al. “Relationshipbetween p53 status and radiosensitivity in human tumour cell lines.”British journal of cancer 73.5 (1996): 581-588.

REFERENCES CITED IN EXAMPLE 13

-   Bolch, W. E., K. F. Eckerman, G. Sgouros, and S. R. Thomas. 2009.    “MIRD Pamphlet No. 21: A Generalized Schema for Radiopharmaceutical    Dosimetry—Standardization of Nomenclature.” Journal of Nuclear    Medicine 50 (3): 477-84. doi:10.2967/jnumed.108.056036.-   Stabin, M G, R B Sparks, and E Crowe. 2005. “OLINDA/EXM: The    Second-Generation Personal Computer Software for Internal Dose    Assessment in Nuclear Medicine.” J Nucl Med 46 (6): 1023-27.

Example 14

Advantages of and Differences when Using Alkylphosphocholine MetalChelates in Place of Radioiodinated Compounds, Such as Those Exemplifiedin Examples 1-4 and 7-10

In this example, we discuss the advantages of using APC metal chelatesinstead of radioiodinated compounds (the compounds exemplified inExamples 1-4 and 7-10). We also discuss factors to be considered by theskilled artisan when optimizing dosages of metal chelates to be used inthe TRT step of the disclosed methods.

Chelates permit the use of a wide variety of stable or radioactive metalions for imaging and therapy. They can be conjugated with a wide varietyof alpha, beta, Auger, gamma and positron emitters whereas iodine islimited to one positron (I-124), one beta (I-131), one gamma (I-123) and1 Auger (I-125) isotope.

Metal Isotopes are Diapeutically More Efficacious than I-131 and I-124.

Lu-177 has fewer high energy gammas which make it more favorable forSPECT imaging and dosimetry. However, its beta energy is slightly lessthan I-131, making it more ideal for treating smaller tumors.

I-131 and Lu-177 are comparable in therapeutic efficacy “horse power”,but there is significantly less contribution to the overall dose fromgamma-emissions for Lu-177. In the case of Y-90, there is negligiblecontribution to the radiation dose from gamma-emissions.

Relative to I-131, Y-90 is more efficacious for killing cancer cells byconventional TRT than I-131, as seen in FIG. 52 and discussed furtherbelow.

The Committee on Medical Internal Radiation Dose (MIRD) developsstandard methods, models, assumptions, and mathematical schema forassessing internal radiation doses from administeredradiopharmaceuticals. The MIRD approach, which simplifies the problem ofassessing radiation dose for many different radionuclides, has beenimplemented in the widely used 510(k) approved software, OLINDA/EXM1.Along with its many standard anthropomorphic phantoms, OLINDA/EXM has aSpheres Model which can be used to approximate tumor doses. The SpheresModel assumes homogeneous distribution of a radiopharmaceutical withinunit-density spheres of a range of tumor masses (0.01-6,000 g).

Using this standard model, we compared Y-90 to 1-131 in terms ofradiation dose normalized by administered radioactivity. The results ofthis comparison, for tumor masses between 1 to 100 g, are displayed inFIG. 52 . Note that the Y-90-to-I-131 ratio reaches 4 for a 4 g tumor,and remains between 4.0 and 4.2 up to a 100 g tumor, strongly suggestingthat on a mCi per mCi basis that Y-90 is between 3.6 and 4.1 times ascytotoxic as I-131 in tumors up to 10 g in size, and about 4.1 timesmore effective in tumors greater than 10 grams in size.

Different Pharmacokinetic Properties

Unlike iodinated analogs, APC chelates are too large to fit into knownalbumin binding pockets in the plasma and therefore exhibit different invivo pharmacokinetic and biodistribution profiles (see FIG. 53 ). Lowerbinding energies lead to larger fractions of free molecule in the plasmawhich affords more rapid tumor uptake. Some APC chelates are cleared viathe renal system, whereas iodinated analogs are eliminated through thehepatobiliary system. APC chelates also accumulate in tumors and clearfrom the blood much quicker than iodinated analog. Faster bloodclearance is directly associated with lower bone marrow and off-targettoxicity of therapeutic radiopharmaceuticals.

These differences in PK and biodistribution profiles lead to differingdose limiting organ toxicity and ultimate utility. Moving fromhematological toxicity to renal or liver for dose limiting toxicitywould increase the utility of radiometal chelates for TRT.

Moreover, the pharmacokinetic profile of the APC chelates can easily bemanipulated by minor changes in the structure of the chelate (e.g.chelate charge). The choice of chelators is vast. Faster clearance fromnormal tissues improves imaging contrast and therapeutic windows,resulting in higher maximum tolerable doses.

APC chelates possess different physico-chemical characteristics thaniodinated analogs. They are much more water-soluble, and therefore donot need surfactants to render them suitable for intravenous injection.APC chelates are based on ionic binding of the metal to the chelate,whereas iodinated compounds form covalent bonds with their carriermolecules. In vivo de-iodination is quite common in alkyl iodideswhereas chelates tend to be extremely stable in vivo.

Once de-iodination occurs, free iodide rapidly accumulates in thethyroid with a very long subsequent excretion half-life, whereas freeradiometals are in general excreted from the body or detoxified muchmore quickly.

In vivo biodistribution of APC chelates can be quite different dependingon the metal ion so the metal and chelate also both contribute to thetumor targeting characteristics of the APC. Not all chelates targettumors. Tumor targeting depends on the cumulative properties of the APCcarrier, the type of chelate (linear chelates undergo rapid renalelimination whereas macrocyclic chelates undergo hepatobiliaryexcretion), and the metal ion. Even slight changes in chelate structureresult in significant variations on the in vivo properties. Simplechanges in isotope can result in changes in tumor targeting larger than50%.

Radioactive APC-metal chelates are easily radiolabeled in nearlyquantitative (>98%) yields under facile conditions, whereasradioiodination yields of iodinated analogs are much lower (typicallyabout 50% for I-131 and 60% for I-124). Moreover, high specificactivities can be achieved with chelates. Synthesis can be done using aradiolabeling kit in any nuclear pharmacy without the requirement ofsophisticated ventilation equipment or training. Radioiodination must bedone in a fume hood fitted with effluent monitoring equipment due to thevolatility of radioactive iodine during the labeling reaction.

Imaging Agents Don't Necessarily Make Good Therapy Agents and ViceVersa.

It cannot be assumed that because there is good tumor uptake with animaging agent that it implies that therapy is obvious. In addition tohaving good tumor uptake, a therapy agent needs to have prolonged tumorretention relative to normal tissues and must be cleared from the bloodquickly in order to lower bone marrow exposure and associated toxicity.Iodinated analogs have prolonged blood residence resulting in doselimiting bone marrow toxicity. In contrast, our APC chelates exhibitmuch faster blood clearance kinetics most likely, as stated above, dueto lower albumin binding in the plasma.

Finally, due to the short path length and physical nature of metallicbeta- and alpha-emitters relative to Iodine-131, there are no exposureconcerns for health care workers or family members following injection.Patients undergoing I-131 therapy often have to be held for some time(up to a week) in a lead shielded room prior to being released from thehospital. Patients injected with radioactive alpha and beta-emitting APCchelates will not be required to remain hospitalized.

Example 15

TRT Delivered by Y90-NM600 in Combination with Administering anAnti-CLA4 Immune Checkpoint Inhibitor Synergistically Inhibits Cancer inan In Vivo Melanoma Model

In this example, we demonstrate the efficacy of the disclosedcombination method, where the in vivo immunization is performed bysystemically administering an immune checkpoint inhibitor (an anti-CTLA4antibody), and the TRT is performed by systemically administering the⁹⁰Y-NM600 chelate used in previous examples.

B78 melanoma subcutaneous flank xenografts were implanted in maleC57BL/6 mice, as described previously. Subsequently, the mice wererandomized to be to be treated with varying doses (25 μCi, 50 μCi, or100 μCi) of ⁹⁰Y-NM600 (Day 1), both with and without anti-CTLA4 antibody(an immune-checkpoint inhibitor) (200 μg on Days 4, 7, and 11) (n=6 foreach experimental group). Both agents were administered by via lateraltail vein injection (i.e., intravenously). Control groups of PBStreatment alone and anti-CTLA4 alone were also included. Tumors weremeasured with calipers twice a week, and animal survival was monitoredfor 60 days.

As shown in FIG. 54 , the three combination therapies(anti-CTLA4+⁹⁰Y-NM600 at three different dosages) showed substantialtumor growth inhibition, as compared to any of the single therapies(anti-CTLA4 or ⁹⁰Y-NM600 alone at three different dosages) or the PBScontrol. After Day 18, combination treatment with 50 or 100 μCi of⁹⁰Y-NM600 with anti-CTLA4 had significantly (p<0.05 by ANOVA) reducedtumor growth compared to PBS, ⁹⁰Y-NM600 alone, or anti-CTLA4 alone. The25 μCi ⁹⁰Y-NM600 combination treatment group with anti-CTLA-4 had anintermediate growth delay response that showed a trend towards doseresponse.

As seen in FIG. 55 , mice treated with 50 μCi of ⁹⁰Y-NM600 combined withanti-CTLA4 exhibited significantly greater aggregate survival than micetreated with TRT alone or PBS vehicle (p<0.05). The log rank was p=0.06for the combination treatment, as compared to anti-CTLA4 alone.

As seen in FIG. 56 , all three combination treatments significantlyimproved survival. Significantly, there were 6/12 (50%) completeresponders in the combination TRT+CTLA4 arms at therapeutic 50 and 100μCi doses of ⁹⁰Y-NM600, as compared to 0/24 complete responders in thenon-combination control arms (PBS, TRT alone at 50 μCi, TRT alone at 100μCi, and anti-CTLA4 alone).

These results illustrate the therapeutic potential of combining the useof a molecular targeted radiotherapeutic agent with any agent thatcauses immune checkpoint inhibition (ICI). The results show that acombination of molecularly targeted TRT and an ICI affords a synergisticeffect, relative to treatment with each agent alone. In addition todemonstrating significant tumor regression, the combined method also hasthe potential to generate immunologic memory and ultimately afford apotent in situ cancer vaccine effect that prevents tumor recurrence.

Conclusion to the Examples

These examples illustrate a novel, never before tested or considered,anti-cancer strategy, based on the synergistic and widely applicablecombination of targeted systemic delivery of radiotherapy with each twodistinct known therapeutic methods: (1) local delivery of combinedimmunotherapy to induce an in situ cancer vaccine, or (2) systemicdelivery of an immunostimulatory agent, such as an immune checkpointinhibitor. As the disclosed metal chelated and radiohalogenatedalkylphosphocholine analogs can target cancers of virtually anyhistology, the local administration of anti-tumor mAb+IL2, or thesystemic administration of immune checkpoint-targeting mAbs or smallmolecules (immune checkpoint inhibitors) finds use for virtually anycancer type (tumor reactive mAbs are approved or in clinical testing fornearly all cancer histological types). Accordingly, the clinicaltranslation of the two different combined strategies have wideapplication for virtually all high risk cancers.

Other embodiments and uses of the invention will be apparent to thoseskilled in the art from consideration from the specification andpractice of the invention disclosed herein. All references cited hereinfor any reason, including all journal citations and U.S./foreign patentsand patent applications, are specifically and entirely incorporatedherein by reference. It is understood that the invention is not confinedto the specific reagents, formulations, reaction conditions, etc.,herein illustrated and described, but embraces such modified formsthereof as come within the scope of the following claims.

1. A method of treating a metastatic cancer in a subject, wherein themetastatic cancer comprises a primary malignant solid tumor and one ormore metastatic tumors capable of causing concomitant immune tolerance,comprising systemically administering to the subject: (a) animmunomodulatory dose of a targeted radiotherapy (TRT) agent that isdifferentially taken up by and retained within the malignant solid tumortissue, wherein the TRT agent is a phospholipid ether metal chelate thathas the formula:

or a salt thereof, wherein: R₁ comprises a chelating agent that ischelated to a metal atom, wherein the metal atom is a metal isotope thatcomprises ⁴⁷Sc, ¹⁷⁷Lu, ¹⁶⁶Ho, ¹⁸⁶Re, ¹⁸⁸Re, ⁶⁷Cu, ¹⁹⁹Au, ¹⁰⁵Rh, ²²³Ra,²²⁵Ac, 212Pb, or ²²⁷Th; a is 0 or 1; Y is selected from the groupconsisting of —H, —OH, —COOH, —COOX, —OCOX, and —OX, wherein X is analkyl or an arylalkyl; and wherein: (i) m is 0, b is 1, n is an integerfrom 12 to 30, and R₂ is —N⁺Z₃, wherein each Z is independently an alkylor an aryl; or (ii) m is 1, b is 1, n is an integer from 12 to 30, andR₂ is —N⁺Z₃, wherein each Z is independently an alkyl or an aryl; or(iii) m is 0, b is 1, n is 18, and R₂ is —N⁺Z₃, wherein each Z isindependently an alkyl or an aryl; or (iv) m is 1, b is 1, n is 18, andR₂ is —N⁺Z₃, wherein each Z is independently an alkyl or an aryl; and(b) one or more immunostimulatory agents capable of stimulating specificimmune cells within the tumor microenvironment; whereby the concomitantimmune tolerance caused by the metastatic tumors is prevented and themetastatic cancer is treated in the subject.
 2. The method of claim 1,wherein the one or more immunostimulatory agents are immune checkpointinhibitors capable of targeting one or more checkpoint molecules.
 3. Themethod of claim 2, wherein the one or more checkpoint molecules that thecheckpoint inhibitors are capable of targeting are selected from thegroup consisting of A2AR (adenosine A2a receptor), BTLA (B and Tlymphocyte attenuator), CTLA4 (cytotoxic T lymphocyte-associated protein4), KIR (killer cell immunoglobulin-like receptor), LAG3 (LymphocyteActivation Gene 3), PD-1 (programmed death receptor 1), PD-L1(programmed death ligand 1), CD40 (cluster of differentiation 40), CD27(cluster of differentiation 27), CD28 (cluster of differentiation 28),CD137 (cluster of differentiation 137), OX40 (CD134; cluster ofdifferentiation 134), OX40L (OX40 ligand; cluster of differentiation252), GITR (glucocorticoid-induced tumor necrosis factorreceptor-related protein), GITRL (glucocorticoid-induced tumor necrosisfactor receptor-related protein ligand), ICOS (inducible T-cellcostimulator), ICOSL (inducible T-cell costimulator ligand), B7H3(CD276; cluster of differentiation 276), B7H4 (VTCN1; V-setdomain-containing T-cell activation inhibitor 1), IDO (Indoleamine2,3-dioxygenase), TIM-3 (T-cell Immunoglobulin domain and Mucin domain3), Gal-9 (galectin-9), and VISTA (V-domain Ig suppressor of T cellactivation).
 4. The method of claim 2, wherein the one or more immunecheckpoint inhibitors comprise one or more anti-immune checkpointmolecule antibodies or one or more small molecule immune checkpointinhibitors that act to block one or more immune checkpoint molecules. 5.The method of claim 4, wherein the one or more anti-immune checkpointmolecule antibodies are selected from the group consisting of ananti-CTLA4 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, ananti-LAG3 antibody, an anti-KIR antibody, an anti-A2AR antibody, andanti-BTLA antibody, an anti-CD40 antibody, an anti-CD27 antibody, ananti-CD28 antibody, an anti-CD137 antibody, an anti-OX40 antibody, ananti-OX40L antibody, a GITR antibody, a GITRL antibody, an ICOSantibody, an ICOSL antibody, a B7H₃ antibody, a B7H₄ antibody, an IDOantibody, a TIM-3 antibody, a Gal-9 antibody, and a VISTA antibody: orwherein the one or more small molecule immune checkpoint inhibitors thatact to block one or more immune checkpoint molecules comprise a smallmolecule PD-L1 inhibitor.
 6. The method of claim 1, wherein theradioactive phospholipid ether metal chelate is NM600.
 7. The method ofclaim 1, wherein the chelating agent is selected from the groupconsisting of 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (D03A)and its derivatives; 1,4,7-triazacyclononane-1,4-diacetic acid (NODA)and its derivatives; 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA)and its derivatives; 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid (DOTA) and its derivatives; 1,4,7-triazacyclononane, 1-glutaricacid-4,7-diacetic acid (NODAGA) and its derivatives;1,4,7,10-tetraazacyclodecane, 1-glutaric acid-4,7,10-triacetic acid(DOTAGA) and its derivatives;1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA) andits derivatives; 1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4,11-diaceticacid (CB-TE2A) and its derivatives; diethylene triamine pentaacetic acid(DTPA), its diester, and its derivatives; 2-cyclohexyl diethylenetriamine pentaacetic acid (CHX-A″-DTPA) and its derivatives;deforoxamine (DFO) and its derivatives;1,2-[[6-carboxypyridin-2-yl]methylamino]ethane (H₂dedpa) and itsderivatives; and DADA and its derivatives, wherein DADA comprises thestructure:


8. The method of claim 1, wherein each Z is independently —CH₂CH₃ or—CH₃.
 9. The method of claim 8, wherein each Z is —CH₃.
 10. The methodof claim 1, wherein the chelating agent chelated to the metal atom isselected from the group consisting of:


11. The method of claim 1, wherein the radioactive phospholipid ethermetal chelate has the formula selected from the group consisting of:

wherein the selected compound is chelated to the metal atom.
 12. Themethod of claim 1, wherein a is 1, b is 1, m is 0, n is 18, and R₂ is—N⁺(CH₃)3.
 13. The method of claim 12, wherein the radioactivephospholipid ether metal chelate is NM600 chelated to the metal atom.14. The method of claim 13, wherein the radioactive phospholipid ethermetal chelate is ¹⁷⁷Lu-NM600.
 15. The method of claim 13, wherein theradioactive phospholipid ether metal chelate is ⁴⁷Sc, ¹⁶⁶Ho-NM600,¹⁸⁶Re-NM600, ¹⁸⁸Re-NM600, ⁶⁷Cu-NM600, ¹⁹⁹Au-NM600, ¹⁰⁵Rh-NM600,²²³Ra-NM600, ²²⁵Ac-NM600, ²¹²Pb-NM600, ²²⁷Th-NM600.
 16. The method ofclaim 1, wherein the TRT agent; the immunostimulatory agent; or both;are administered intravenously.
 17. The method of claim 1, wherein thesubject is a human.
 18. The method of claim 1, wherein the cancer thatis treated is selected from the group consisting of melanoma,neuroblastoma, lung cancer, adrenal cancer, colon cancer, colorectalcancer, ovarian cancer, prostate cancer, liver cancer, subcutaneouscancer, squamous cell cancer of the skin or head or neck, intestinalcancer, retinoblastoma, cervical cancer, glioma, breast cancer,pancreatic cancer, soft tissue sarcoma, Ewings sarcoma,rhabdomyosarcoma, osteosarcoma, Wilms' tumor, and pediatric braintumors.
 19. The method of claim 1, wherein the cancer is treated withoutadministering to the subject an antibody to a tumor antigen that is nota checkpoint molecule or an anti-GD2 antibody.
 20. The method of claim1, wherein the immunomodulatory dose of the targeted radiotherapy (TRT)agent delivers a radiation dose of from 2 Gy to 8 Gy to the metastatictumors.